Internal transcribed spacer primer evaluation for vascular plant metabarcoding

The unprecedented ongoing biodiversity decline necessitates scalable means of monitoring in order to fully understand the underlying causes. DNA metabarcoding has the potential to provide a powerful tool for accurate and rapid biodiversity monitoring. Unfortunately, in many cases, a lack of universal standards undermines the widespread application of metabarcoding. One of the most important considerations in metabarcoding of plants, aside from selecting a potent barcode marker, is primer choice. Our study evaluates published ITS primers in silico and in vitro, through mock communities and presents newly designed primers. We were able to show that a large proportion of previously available ITS primers have unfavourable attributes. Our combined results support the recommendation of the introduced primers ITS-3p62plF1 and ITS-4unR1 as the best current universal plant specific ITS2 primer combination. We also found that PCR optimisation, such as the addition of 5% DMSO, is essential to obtain meaningful results in ITS2 metabarcoding. Finally, we conclude that continuous quality assurance is indispensable for reliable metabarcoding results.

First Report of Alternaria arborescens Causing Early Blight on Tomato in Iraq

In this paper, the isolation of the fungal species Alternaria arborescens was done from symptomatic tomato leaves and diagnosed morphologically and molecularly using ITS primers. Subsequently, pathogenicity determination was achieved for the diagnosed fungal species on tomato plant. It’s noteworthy, this work on A. arborescens was not previously recorded as a potential pathogen on the shoot system of tomato in Iraq.

Towards Managing and Controlling Aflatoxin Producers Within Aspergillus Species in Infested Rice Grains Collected from Local Markets in Kenya

Rice grains can be attacked by a range of pathogens, including Aspergillus species, which can cause the accumulation of aflatoxins and represent a serious threat to the consumers. Aflatoxins are secondary metabolites synthesized by Aspergillus species and naturally occur in various foodstuffs. In this study, we sought to analyze the prevalence of aflatoxin-producing Aspergillus spp. in rice grains currently sold in Kenyan local markets. We analyzed a total of 98 samples randomly collected and primarily analyzed to observe moisture content and fungal growth. We then isolated Aspergillus species, characterized them morphologically and using the Internal transcribed spacer (ITS) primers. Finally, we screened them for aflatoxin-producing isolates targeting Norsolorinic Acid (nor-1) and Versicolorin (ver-1) specific genes involved in aflatoxin biosynthesis. We observed that all tested samples were contaminated. The highest prevalence of Aspergillus species and aflatoxigenic fungal species, had values of 66% and 36.4% for nor-1 and ver-1, respectively. In total, 66% of all isolates were confirmed to be aflatoxin producers. The occurrence of high contamination levels of Aspergillus species points to the possibility of production of aflatoxins in rice grains. This work provides a baseline for future studies on the occurrence of mycotoxigenic fungal species in rice grains being sold in local markets and strategies to control these aflatoxigenic strains at pre- and post-harvest levels.

Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

ABSTRACT We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for maintaining currently recommended microbiome research techniques as the state of the art. Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies. IMPORTANCE We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for maintaining currently recommended microbiome research techniques as the state of the art.