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 .