DISCUSSION
This study offers a rapid method for the detection of N. barbataeDNA from cultured and field collected samples. Diagnostic confirmation had previously relied on cultivation and subsequent metabarcoding analysis to confirm presence of Nannizziopsis infection (Peterson et al., 2020) leading to lengthy delays in obtaining results due to the slow growth characteristics of these species (Paré & Sigler, 2016).
Our qPCR assay was able to correctly distinguish DNA samples of N. barbatae from five other species of Nannizziopsis . The assay was sufficiently sensitive to detect the presence of less than one genomic equivalent per reaction, owing to the presence of multiple copies of the mitogenome for every nuclear genome. We expect there to be an average of between six to nine mitochondrial genome copies per nuclear genome for the N. barbatae isolates sequenced in this study based on differences observed in the amount of sequencing read coverage between the nuclear and mitochondrial genome. However, we caution that this may be highly variable and with only a few isolates sequenced to date, the use of higher limit of detection should be considered until sufficient data on copy number can be attained.
We also demonstrate the utility of this assay to screen a free-living population of infected reptiles. In addition to the 67 samples that tested positive for infection with N. barbatae , there were seven instances of what appear to be false negative results out of 74 visibly diseased animals from this screening set. As these occurred only in the mildly diseased animals (rating 1 or 2, Supplementary Table 1), we posit that this is likely the result of the state of the infected site being small and difficult to recover fungal DNA, and/or variation in the swab sampling techniques from different handlers that yielded lower sample material. There were no false positive results observed in this sample set suggesting no contaminating sources of non-target DNA were encountered.
We report that the high similarity between the species N. vriesiiand N. dermatitidis extends across the entire mitochondrial genome with the notable exception of an intron present in thenad1 gene in N. dermatitidis . This high level of similarity suggests some taxonomic revision may be appropriate to either group these two species together or split the two strains ofN. barbatae . The varying occurrence of introns was also observed between the two strains of N. barbatae in this study isolated from infected reptiles approximately 10 years apart. Each of these introns was found to contain either an LAGLI-DADG or GIY-YIG endonuclease domain motif. These domains encode homing nucleases, suggesting these introns possess a capacity for self-splicing (Megarioti & Kouvelis, 2020). Intra-specific variations in the presence of mitochondrial introns has been reported for other species of fungi (Deng et al., 2020; Freel, Friedrich, Hou, & Schacherer, 2014) and the non-uniform inclusion of introns in the mitochondrial genes found in these closely related species may indicate the activity of mobile elements during their evolutionary history. An intron is present in thenad1 gene of all Nannizziopsis andParanannizziopsis isolates included in this study with the exception of N. vriesii (UAMH 3527) which was originally isolated in 1972, two decades earlier than any of the other fungal species. This strain of N. vriesii was also found in Europe, quite distant from each of the other strains that were found in either Australia or North America. For each N. barbatae isolate, this intron is 1,047 bp in length and was dissimilar in only two positions, which are outside of the assay target region, despite UAMH 1185 being isolated from its reptile host over 10 years prior and from a geographic location separated by more than 700 km. Even though the nad1 gene intron contains an GIY-YIG motif, it is conserved in all but one species ofNannizziopsis included in this study and could be amplified using the same primer pair in each case. We believe this DNA target is stable enough for further applications using this assay for the detection ofN. barbatae .