Development and validation of TOF/Q-TOF MS/MS, HPLC method and in vitro bio-strategy for aflatoxin mitigation.

Some secondary metabolites produced by fungi are carcinogenic, hepatotoxic, and/or cause birth defects in humans and animals. We developed and optimised bio-analytical tools for detection of metabolites, aflatoxins and evaluated the effectiveness of the methods in co-infected maize tissues. Isolate KSM012 (atoxigenic) demonstrated no peaks and no blue fluorescence on HPLC and TLC plates respectively confirming non-toxicity. AFB1 and AFB2 were produced by Isolate KSM015 in addition to AFG1 and AFG2, which is an indication of possible SBG morphotype. The limits of quantification and detection ranged from 0.02 to 35.81 µg/mL and 0.01-6.8 µg/mL, respectively. The best mass spectrum with lowest noise was obtained at 100% ACN and sterile water spiked with 0.1% formic acid at a flow rate of 0.3 mL/min. The positive ion mode with electrospray ionisation application exhibited better fragmentation for mycotoxins. In total 17 metabolites were detected by targeted and formula mass. KDVI maize line exhibited high fungal colonisation in comparison to GAF4 at equal co-infection ratio 50:50. AFB1 and AFG2 were remarkably higher in GAF4 in comparison to sensitive KDV1 (p ˂ 0.05). The detection limits, linearity and sensitivity showed the method developed was suitable for the determination of mycotoxin in comparisons to the guidelines of European Commission 657/EC 2002.

Molecular and Vegetative Compatibility Groups Characterization of Aspergillus flavus Isolates from Kenya.

The genus Aspergillus contains diverse species and the identification is complicated. Vegetative compatibility groups (VCGs) and molecular mechanisms were deployed to study the species. The study was randomly conducted in four counties in Kenya based on the history of aflatoxicosis and maize cultivation. Thirty-seven Aspergillus flavus isolates from Nandi, Kisumu, Homa Bay and Makueni were characterized to determine their taxonomic status based on their VCGs and genotypes. A phylogenetic analysis of ITS1 and ITS2 sequences of the isolates investigated revealed ITS primers discriminating some of the A. flavus isolates as 100% sequence identity to the RefSeq. Nit mutants' complementation test revealed strong heterokaryon incompatibility between isolates of Nandi region (67%) and Makueni (33%). The trend based on VCGs and molecular findings showed high incidence of toxigenic A. flavus in Makueni, which could be the reason why the region frequently experiences chronic aflatoxicosis incidences over the last few decades as compared to other regions. Interestingly, we have discovered all S and L-morphotypes including the rare S/L-morphotypes, which implies that Kenya is home to all morphotypes of A. flavus. Thus, the analysis provides a deeper understanding of the taxonomic relationship between the A. flavus isolates and could help contextualise the data obtained for each isolate with respect to VCG genetic diversity and genotypes. Determining the primary causal agents of aflatoxin contamination is critical for predicting risk of contamination events and designing and implementing effective management strategies.

Breakthroughs in the discovery and use of different peroxidase isoforms of microbial origin.

Peroxidases are classified as oxidoreductases and are the second largest class of enzymes applied in biotechnological processes. These enzymes are used to catalyze various oxidative reactions using hydrogen peroxide and other substrates as electron donors. They are isolated from various sources such as plants, animals and microbes. Peroxidase enzymes have versatile applications in bioenergy, bioremediation, dye decolorization, humic acid degradation, paper and pulp, and textile industries. Besides, peroxidases from different sources have unique abilities to degrade a broad range of environmental pollutants such as petroleum hydrocarbons, dioxins, industrial dye effluents, herbicides and pesticides. Ironically, unlike most biological catalysts, the function of peroxidases varies according to their source. For instance, manganese peroxidase (MnP) of fungal origin is widely used for depolymerization and demethylation of lignin and bleaching of pulp. While, horseradish peroxidase of plant origin is used for removal of phenols and aromatic amines from waste waters. Microbial enzymes are believed to be more stable than enzymes of plant or animal origin. Thus, making microbially-derived peroxidases a well-sought-after biocatalysts for versatile industrial and environmental applications. Therefore, the current review article highlights on the recent breakthroughs in the discovery and use of peroxidase isoforms of microbial origin at a possible depth.

Correction: Mitema, A. et al. The Development of a qPCR Assay to Measure Aspergillus flavus Biomass in Maize and the Use of a Biocontrol Strategy to Limit Aflatoxin Production. Toxins 2019, 11, 179.

The authors wish to make the following correction to their paper [...].

The Development of a qPCR Assay to Measure Aspergillus flavus Biomass in Maize and the Use of a Biocontrol Strategy to Limit Aflatoxin Production.

Aspergillus flavus colonisation of maize can produce mycotoxins that are detrimental to both human and animal health. Screening of maize lines, resistant to A. flavus infection, together with a biocontrol strategy, could help minimize subsequent aflatoxin contamination. We developed a qPCR assay to measure A. flavus biomass and showed that two African maize lines, GAF4 and KDV1, had different fungal loads for the aflatoxigenic isolate (KSM014), fourteen days after infection. The qPCR assay revealed no significant variation in A. flavus biomass between diseased and non-diseased maize tissues for GAF4, while KDV1 had a significantly higher A. flavus biomass (p < 0.05) in infected shoots and roots compared to the control. The biocontrol strategy using an atoxigenic isolate (KSM012) against the toxigenic isolate (KSM014), showed aflatoxin production inhibition at the co-infection ratio, 50:50 for both maize lines (KDV1 > 99.7% and GAF ≥ 69.4%), as confirmed by bioanalytical techniques. As far as we are aware, this is the first report in Kenya where the biomass of A. flavus from maize tissue was detected and quantified using a qPCR assay. Our results suggest that maize lines, which have adequate resistance to A. flavus, together with the appropriate biocontrol strategy, could limit outbreaks of aflatoxicoses.

Vegetative compatibility and phenotypic characterization as a means of determining genetic diversity of Aspergillus flavus isolates.

Toxigenic Aspergillus species produce mycotoxins that are carcinogenic, hepatotoxic and teratogenic immunosuppressing agents in both human and animals. Kenya frequently experiences outbreaks of aflatoxicosis with the worst occurring in 2010, which resulted in 215 deaths. We examined the possible reasons for these frequent aflatoxicosis outbreaks in Kenya by studying Aspergillus flavus diversity, phenotypes and mycotoxin profiles across various agricultural regions. Using diagonal transect random sampling, maize kernels were collected from Makueni, Homa Bay, Nandi, and Kisumu counties. Out of 37 isolates, nitrate non-utilizing auxotrophs complementation test revealed 20 vegetative compatibility groups. We designated these groups by the prefix "KVCG", where "K" represented Kenya and consequently assigned numbers 1-20 based on our findings. KVCG14 and KVCG15 had highest distribution frequency (n = 13; 10.8 %). The distribution of the L-, S- and S-/L-morphotypes across the regions were 57 % (n = 21); 7 % (n = 3) and 36 % (n = 13), respectively. Furthermore, a unique isolate (KSM015) was identified that had characteristics of S-morphotype, but produced both aflatoxins B and G. Coconut agar medium (CAM) assay, TLC and HPLC analyses confirmed the presence or absence of aflatoxins in selected toxigenic and atoxigenic isolates. Diversity index (H') analyses ranged from 0.11 (Nandi samples) to 0.32 (Kisumu samples). Heterokaryon compatibility ranged from 33 % (for the Makueni samples, n = 3) to 67 % (Nandi samples, n = 6). To our knowledge, this is the first reported findings for A. flavus diversity and distribution in Nandi, Homa Bay and Kisumu counties and may assist current and future researchers in the selection of biocontrol strategies to mitigate aflatoxin contamination as has been researched in Makueni and neighbouring counties.