Current metabarcoding studies aiming to characterize microbial communities generally rely on the amplification and sequencing of relatively short DNA regions. For fungi, the internal transcribed spacer (ITS) region in the ribosomal RNA (rRNA) operon has been accepted as the formal fungal barcode. Despite an increasing number of fungal metabarcoding studies, the amplification efficiency of primers is generally not tested prior to their application in metabarcoding studies. Some of the challenges that metabarcoding primers should overcome efficiently are the amplification of target DNA strands in samples rich in non-target DNA and environmental pollutants, such as humic acids, that may have been co-extracted with DNA. In the current study, three selected primer pairs were tested for their suitability as fungal metabarcoding primers. The selected primer pairs include two primer pairs that have been frequently used in fungal metabarcoding studies (ITS1F/ITS2 and ITS3/ITS4) and a primer pair (ITS86F/ITS4) that has been shown to efficiently amplify the ITS2 region of a broad range of fungal taxa in environmental soil samples. The selected primer pairs were evaluated in a 454 amplicon pyrosequencing experiment, real-time PCR (qPCR) experiments and in silico analyses. Results indicate that experimental evaluation of primers provides valuable information that could aid in the selection of suitable primers for fungal metabarcoding studies. Furthermore, we show that the ITS86F/ITS4 primer pair outperforms other primer pairs tested in terms of in silico primer efficiency, PCR efficiency, coverage, number of reads and number of species-level operational taxonomic units (OTUs) obtained. These traits push the ITS86F/ITS4 primer pair forward as highly suitable for studying fungal diversity and community structures using DNA metabarcoding.

Seven banana cultivars (Musa acuminata, AAA group) were inoculated with two species of vesicular arbuscular mycorrhizal (VAM) fungi (Glomus mosseae and Glomus macrocarpum) in a greenhouse experiment. Inoculated plants had generally greater shoot dry weight and shoot phosphorus concentrations compared to the noninoculated plants. A great variation in dependency on mycorrhizal colonization was observed among the banana cultivars. Cv. Williams showed the highest relative mycorrhizal dependency (RMD) and cv. Poyo the lowest. For all the cultivars studied, inoculation with G. macrocarpum resulted in the highest RMD values. Both root dry weight and root hair length or density of the noninoculated plants were inverserly correlated with the RMD values of cultivars.

Summary• A new method is described for monitoring hyphal 32 P transport in compartmented, monoxenic mycorrhizal root cultures. Nondestructive time-course measurements of P transport in hyphae were obtained by capturing digital autoradiograms on P-imaging screens, and comparing with growth observed by optical scanning. 32 P distribution measured by densitometry on the day of harvest closely agreed with values obtained by liquid scintillation counting after destructive harvest.• Virtually all labeled PO 4 was absorbed by arbuscular mycorrhizal (AM) hyphae, but transfer to the roots appeared to be incomplete. P transport was not unidirectional towards the roots, as 32 P was also transported from the root compartment to the hyphal compartment. Net P flux rates were calculated for hyphae crossing between compartments, taking bidirectional flow into account.• Amounts of transported P were poorly correlated with extra-radical hyphal length and root d. wt, but highly correlated with the number of hyphae crossing the barrier separating the two compartments. Such correlations were highest when only hyphae with detectable protoplasmic streaming were considered.• The method was tested using radiolabeled P sources, H 2 PO 4 -and cytidine triphosphate (CTP), and the AM fungi, Glomus intraradices and G. proliferum. Fungal transport of 32 P from CTP was much slower than from PO 4 for both fungi.

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