Detection of Mycoviruses - flavus populations and their mycovirome

Molecular tools for the characterization of Aspergillus flavus strains

A. flavus populations and their mycovirome

1. Background

1.3. Detection of Mycoviruses

To identificate mycoviruses, their genomes must be fully sequenced and annotated. This information is crucial for assessing the diversity of the virome in Aspergillus species, since the electrophoretic profiles of the dsRNA elements on agarose gels cannot be reliably used to infer evolutionary relationships with other viruses. Nerva et al in their work of 2015 reported how detection techniques evolved over time, from electrophoretic gel to NGS techonolgy. The classic method for the detection of mycoviruses consists of the purification of dsRNA using CF11 cellulose;

this simple and cheap technique is able to detect the most of viruses with dsRNA genomes (and, with some extent, also dsRNA replication intermediates from ssRNA genome viruses), and this is probably the reason why the majority of the so far described mycoviruses have a dsRNA genome (Nerva et al. 2015). Another common way of detection is through virus particle’s purification using

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differential centrifugation protocols combined with observation by transmission electron microscopy (TEM). This approach allows the detection of viruses with different kind of genomes, but requires a traditional virological expertise and expensive equipment, which are not always available in the most common laboratories working on fungal and molecular biology; in addition, this technique is unsuitable for “naked” viruses, a fairly common feature for mycoviruses. The identification of new mycovirus taxonomic groups therefore strongly requires new approaches of diagnosis and detection. In this sense, a new impulse to this research field came from the even more diffused availability of next-generation sequencing (NGS) dedicated protocols; in fact, as reported in a wide number of publications, NGS techniques proved di be able to detect the presence of viral sequences in samples with very low viral titer. For example, Al Rwahnih et al.,(2011) and Espach, (2013) analyzed the total RNA extract from plant using NGS techniques, reaching not only to detect the plant viruses, but also to characterize at the same time the whole virome of the fungal endophytes. If applied to total RNA extract, NGS allows the detection of all the different viral taxa because, independently from the DNA or RNA genome, all of them must produce mRNA during their replication cycle. NGS can be applied also to small RNA (sRNA) libraries: Kreuze et al. (2009) showed the possibility to assemble new and unknown viral sequences from RNA libraries obtained from plants. To our knowledge, this approach has been used very recently for de novo assembly of viral genomes from fungi (Vainio et al., 2015). Nerva et al (2015) reported a complete method of RNAseq.

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2. Definition of the mycovirome inside A. flavus population from Italy and Madagascar A selection of 62 A. flavus strains, isolated from maize grains collected in the Po valley (35 strains from Emilia-Romagna, 9 from Friuli-Venezia Giulia, 6 from Piemonte, 6 from Veneto, 3 from Lombardia and 3 from Trentino Alto-Adige), and 36 A. flavus strains and 5 strains of A. tamarii from maize grains collected in the Lokobe nature reserve in Madagascar, was subjected to the analysis of the mycovirome (Figure 91).

Figure 91: Complete list of Italian and Madagascar strains.

Strains were grown in YES 5% medium in multi-well plates; the total RNA was extracted from single mycelia before conidiation, then put together at the same concentration (1ng) for each sample. RNA mix was subjected to RNAseq analysis. As results of sequence alignment in specific databases, viral dsRNAs have been identified. The presence of strain-specific viruses was assessed in each single

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strain subjecting the relevant RNA to reverse transcription and then to Real Time PCR analysis.

Tables 11 and 12 report the complete list of A. flavus strains with the relevant mycoviruses.

MADAGASCAR

STRAIN MYCOVIRUS STRAIN MYCOVIRUS

LOK 16 - Aspergillus flavus negative single-stranded RNA 1

Aspergillus flavus negative single-stranded RNA 2 LOK 62 - Aspergillus flavus negative single-stranded RNA 1 Aspergillus flavus negative single-stranded RNA 2

LOK 18 - Aspergillus flavus negative single-stranded RNA 1

Aspergillus flavus negative single-stranded RNA 2 LOK 64 -