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Page 10 of 19 Kok et al. Microbiome Res Rep 2023;2:30 https://dx.doi.org/10.20517/mrr.2023.16

Figure 4. Phage discovery (A) Schematic of phage enrichment and isolation process (created with BioRender.com); (B) Positive spot
tests after enrichment; (C) Representative TEM image of Phage Lilo (Harrison cluster); (D) Representative TEM image of Phage Ollie
(Vegas cluster). Scale bars = 50 nm. TEM: Transmission electron microscopy.

Isolating phages from soil/hive material
The 430 samples received were processed using an enrichment technique followed by three rounds of
plaque purification [Figure 4A and B]. The plaque morphologies of all isolated phages were tiny, pin-prick
plaques that appeared clear. When tested by a standard spot titer plate method, all of our phages had very
low effective titers ranging from 6.7 × 10 to 2.7 × 10 . A RAMP-UP technique was used to increase the titer
2
5
of all phages in order to extract DNA and send it for sequencing . Once these titers were raised by this
[30]
8
method to > 1 × 10 , we proceeded with visualization and complete genome sequencing.
Transmission electron microscopy of isolated phage
Electron microscopy was undertaken on Phage Lilo (Harrison Cluster) Figure 4C and Phage Ollie (Vegas
Cluster) [Figure 4D]. These two phages were selected as they each represented one of the two clusters of
phages found in New Zealand. These revealed phages with long, filamentous, non-contractile tails; phages
with these types of tails are classified as having Siphoviridae morphology . All the new phages reported
[53]
here are Gochnauervirinae, a subfamily in the Caudoviricetes class. All but one of the known P. larvae
phages have this morphotype . Phage Lilo had a tail of approximately 148 nm in length, with a prolate
[19]
head measuring approximately 105 nm by 41 nm. Phage Ollie had a tail approximately 156 nm in length,
with a prolate head measuring approximately 106 nm by 43 nm.

Host range testing
Specificity of the New Zealand isolated phages on each of the eight native P. larvae isolates identified in this
paper, as well as 22 native P. larvae isolates provided by the ApiWellbeing team was carried out using
[54]
standard spot test assays. Phages were scored as positive or negative for cell lysis. Nine distinct infection
patterns were identified [Figure 5].