Phenotypic characterization of phage vB_vcM_Kuja.

Bacteriophage therapy targeting the increasingly resistant Vibrio cholerae is highly needed. Hence, studying the phenotypic behavior of potential phages under different conditions is a prerequisite to delivering the phage in an active infective form. The objective of this study was to characterize phage VP4 (vB_vcM_Kuja), an environmental vibriophage isolated from River Kuja in Migori County, Kenya in 2015. The phenotypic characteristics of the phage were determined using a one-step growth curve, restriction digestion profile, pH, and temperature stability tests. The results revealed that the phage is stable through a wide range of temperatures (20-50°C) and maintains its plaque-forming ability at pH ranging from 6 to 12. The one-step growth curve showed a latent period falling between 40 and 60 min, while burst size ranged from 23 to 30 plaque-forming units/10 µl at the same host strain. The restriction digestion pattern using EcoRI, SalI, HindIII, and XhoI enzymes showed that HindIII could cut the phage genome. The phage DNA could not be restricted by the other three enzymes. The findings of this study can be used in future studies to determine phage-host interactions.

Aflatoxin contamination of maize and groundnut in Burundi: Distribution of contamination, identification of causal agents and potential biocontrol genotypes of Aspergillus flavus.

Aflatoxin contamination of the staples maize and groundnut is a concern for health and economic impacts across sub-Saharan Africa. The current study (i) determined aflatoxin levels in maize and groundnut collected at harvest in Burundi, (ii) characterized populations of Aspergillus section Flavi associated with the two crops, and (iii) assessed aflatoxin-producing potentials among the recovered fungi. A total of 120 groundnut and 380 maize samples were collected at harvest from eight and 16 provinces, respectively. Most of the groundnut (93%) and maize (87%) contained aflatoxin below the European Union threshold, 4 µg/kg. Morphological characterization of the recovered Aspergillus section Flavi fungi revealed that the L-morphotype of A. flavus was the predominant species. Aflatoxin production potentials of the L-morphotype isolates were evaluated in maize fermentations. Some isolates produced over 137,000 µg/kg aflatoxin B1. Thus, despite the relatively low aflatoxin levels at harvest, the association of both crops with highly toxigenic fungi poses significant risk of post-harvest aflatoxin contamination and suggests measures to mitigate aflatoxin contamination in Burundi should be developed. Over 55% of the L-morphotype A. flavus did not produce aflatoxins. These atoxigenic L-morphotype fungi were characterized using molecular markers. Several atoxigenic genotypes were detected across the country and could be used as biocontrol agents. The results from the current study hold promise for developing aflatoxin management strategies centered on biocontrol for use in Burundi to reduce aflatoxin contamination throughout the value chain.

Susceptibility of the Western Honey Bee Apis mellifera and the African Stingless Bee Meliponula ferruginea (Hymenoptera: Apidae) to the Entomopathogenic Fungi Metarhizium anisopliae and Beauveria bassiana.

This study assessed the nontarget effect of entomopathogenic fungi on the Western honey bee Apis mellifera L. and the African stingless bee Meliponula ferruginea Cockrell (Hymenoptera: Apidae). Pathogenicity of five Metarhizium anisopliae (ICIPE 7, ICIPE 20, ICIPE 62, ICIPE 69, and ICIPE 78) (Metschnikoff) Sorokin (Hypocreales: Clavicipitaceae) and one of Beauveria bassiana (ICIPE 284) (Balsamo) Vuillemin (Hypocreales: Cordicipitaceae) isolates were evaluated on bees at 108 conidia/ml. Conidial acquisition was evaluated immediately after exposure. Apis mellifera acquired more conidia (2.8 × 104-1.3 × 105 conidia per bee) compared to M. ferruginea (1.1 × 104-2.3 × 104 conidia per bee). In the bioassay with A. mellifera, ICIPE 7, ICIPE 20, and ICIPE 69 moderately reduced the survival by 16.9, 17.4, 15.3%, with lethal times LT10 = 7.4, 7.6, 8.1 d and LT25 = 8.7, 10.0, 9.9 d, respectively. The three isolates caused A. mellifera mycosis of 11.6-18.5%. None of the isolates had a significant effect on M. ferruginea. The tested isolates are nontoxic to bees according to the International Organization of Biological Control (IOBC) classification. However, the effect of ICIPE 7, ICIPE 20, and ICIPE 69 merits further studies on bee colonies, especially those of A. mellifera, under field conditions.

Aflatoxin-producing fungi associated with pre-harvest maize contamination in Uganda.

Maize is an important staple crop for the majority of the population in Uganda. However, in tropical and subtropical climates, maize is frequently contaminated with aflatoxins, a group of cancer-causing and immuno-suppressive mycotoxins produced by Aspergillus section Flavi fungi. In Uganda, there is limited knowledge about the causal agents of aflatoxin contamination. The current study determined both the aflatoxin levels in pre-harvest maize across Uganda and the structures of communities of aflatoxin-producing fungi associated with the maize. A total of 256 pre-harvest maize samples were collected from 23 major maize-growing districts in eight agro-ecological zones (AEZ). Maize aflatoxin content ranged from 0 to 3760 ng/g although only around 5% for Ugandan thresholds. For EU it is about 16% of the samples contained aflatoxin concentrations above tolerance thresholds. A total of 3105 Aspergillus section Flavi isolates were recovered and these were dominated by the A. flavus L morphotype (89.4%). Densities of aflatoxin-producing fungi were negatively correlated with elevation. Farming systems and climatic conditions of the AEZ are thought to have influenced communities' structure composition. Fungi from different AEZ varied significantly in aflatoxin-producing abilities and several atoxigenic genotypes were identified. The extremely high aflatoxin concentrations detected in some of the studied regions indicate that management strategies should be urgently designed for use at the pre-harvest stage. Atoxigenic genotypes detected across Uganda could serve as aflatoxin biocontrol agents to reduce crop contamination from fields conditions and throughout the maize value chain.

Changes in the Fungal Microbiome of Maize During Hermetic Storage in the United States and Kenya.

Prior to harvest, maize kernels are invaded by a diverse population of fungal organisms that comprise the microbiome of the grain mass. Poor post-harvest practices and improper drying can lead to the growth of mycotoxigenic storage fungi and deterioration of grain quality. Hermetic storage bags are a low-cost technology for the preservation of grain during storage, which has seen significant adoption in many regions of Sub-Saharan Africa. This study explored the use of high-throughput DNA sequencing of the fungal Internal Transcribed Spacer 2 (ITS2) region for characterization of the fungal microbiome before and after 3 months of storage in hermetic and non-hermetic (woven) bags in the United States and Kenya. Analysis of 1,377,221 and 3,633,944 ITS2 sequences from the United States and Kenya, respectively, resulted in 251 and 164 operational taxonomic units (OTUs). Taxonomic assignment of these OTUs revealed 63 and 34 fungal genera in the US and Kenya samples, respectively, many of which were not detected by traditional plating methods. The most abundant genus was Fusarium, which was identified in all samples. Storage fungi were detected in the grain mass prior to the storage experiments and increased in relative abundance within the woven bags. The results also indicate that storage location had no effect on the fungal microbiome of grain stored in the United States, while storage bag type led to significant changes in fungal composition. The fungal microbiome of the Kenya grain also underwent significant changes in composition during storage and fungal diversity increased during storage regardless of bag type. Our results indicated that extraction of DNA from ground kernels is sufficient for identifying the fungi associated with the maize. The results also indicated that bag type was the most important factor influencing changes in fungal microbiome during storage. The results also support the recommended use of hermetic storage for reducing food safety risks, especially from mycotoxigenic fungi.

Antifungal Activity of Essential Oil of Eucalyptus camaldulensis Dehnh. against Selected Fusarium spp.

The objective of this study was to evaluate the antifungal activity of essential oil (EO) of Eucalyptus camaldulensis Dehnh. against five Fusarium spp. commonly associated with maize. The essential oil had been extracted by steam distillation in a modified Clevenger-type apparatus from leaves of E. camaldulensis and their chemical composition characterized by gas chromatography mass spectrometry. Poisoned food technique was used to determine the percentage inhibition of mycelial growth, minimum inhibitory concentration, and minimum fungicidal concentration of the EO on the test pathogens. Antifungal activity of different concentrations of the EO was evaluated using disc diffusion method. The most abundant compounds identified in the EO were 1,8-cineole (16.2%), α-pinene (15.6%), α-phellandrene (10.0%), and p-cymene (8.1%). The EO produced complete mycelial growth inhibition in all the test pathogens at a concentration of 7-8 µL/mL after five days of incubation. The minimum inhibitory concentration and minimum fungicidal concentration of the EO on the test fungi were in the range of 7-8 µL/mL and 8-10 µL/mL, respectively. These findings confirm the fungicidal properties of E. camaldulensis essential oils and their potential use in the management of economically important Fusarium spp. and as possible alternatives to synthetic fungicides.

Morphological and Genetic Diversity of Rhizobia Nodulating Cowpea (Vigna unguiculata L.) from Agricultural Soils of Lower Eastern Kenya.

Limited nitrogen (N) content in the soil is a major challenge to sustainable and high crop production in many developing countries. The nitrogen fixing symbiosis of legumes with rhizobia plays an important role in supplying sufficient N for legumes and subsequent nonleguminous crops. To identify rhizobia strains which are suitable for bioinoculant production, characterization of rhizobia is a prerequisite. The objective of this study was to assess the morphological and genetic diversity of rhizobia that nodulates cowpea in agricultural soils of lower eastern Kenya. Twenty-eight rhizobia isolates were recovered from soil samples collected from farmers' fields in Machakos, Makueni, and Kitui counties in lower eastern Kenya and characterized based on morphological characteristics. Thirteen representative isolates were selected and characterized using BOX repetitive element PCR fingerprinting. Based on the dendrogram generated from morphological characteristics, the test isolates were distributed into two major clusters at a similarity of 75%. Phylogenetic tree, based on BOX repetitive element PCR, grouped the isolates into two clusters at 90% similarity level. The clustering of the isolates did not show a relationship to the origin of soil samples, although the isolates were genetically diverse. This study is a prerequisite to the selection of suitable cowpea rhizobia to develop bioinoculants for sustainable crop production in Kenya.

Chemical Composition and Antibacterial Activity of Essential Oils of Tagetes minuta (Asteraceae) against Selected Plant Pathogenic Bacteria.

The objective of this study was to determine the chemical composition and antibacterial activity of essential oils (EOs) of Tagetes minuta against three phytopathogenic bacteria Pseudomonas savastanoi pv. phaseolicola, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas axonopodis pv. manihotis. The essential oils were extracted using steam distillation method in a modified Clevenger-type apparatus while antibacterial activity of the EOs was evaluated by disc diffusion method. Gas chromatography coupled to mass spectrometry (GC/MS) was used for analysis of the chemical profile of the EOs. Twenty compounds corresponding to 96% of the total essential oils were identified with 70% and 30% of the identified components being monoterpenes and sesquiterpenes, respectively. The essential oils of T. minuta revealed promising antibacterial activities against the test pathogens with Pseudomonas savastanoi pv. phaseolicola being the most susceptible with mean inhibition zone diameters of 41.83 and 44.83 mm after 24 and 48 hours, respectively. The minimum inhibitory concentrations and minimum bactericidal concentrations of the EOs on the test bacteria were in the ranges of 24-48 mg/mL and 95-190 mg/mL, respectively. These findings provide a scientific basis for the use of T. minuta essential oils as a botanical pesticide for management of phytopathogenic bacteria.