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First report of branch cankers caused by Sphaerulina musiva on Populus purdomii in the 1
United States 2
C. Newman, A. L. Loyd, and J. A. Smith, University of Florida, School of Forest Resources and 3
Conservation, Gainesville, FL 32611 4
In November of 2017 numerous branch cankers were observed on a specimen of Populus 5
purdomii in a cultivated plant collection in Boone County, Kentucky. Populus purdomii is native 6
to China, and is in the section Tacamahaca in the genus Populus, which also includes the 7
important forestry species P. trichocarpa. Septoria canker caused by Sphaerulina musiva is one 8
of the most destructive diseases of introduced and hybrid poplars (Bier 1939). Branch cankers, 9
typical of those caused by S. musiva were abundant on small to medium-sized branches. Cankers 10
displayed sunken and discolored tissue with swollen margins, and were causing dieback in the 11
canopy. Isolations were made from six, 1 mm
3
excised pieces of tissue from the transition zone 12
of three diseased branches, surface sterilized in 5% sodium hypochlorite for 30 seconds, and 13
placed onto quarter-strength acidified potato dextrose agar. Isolate AL-PC5 has been deposited 14
into the USDA fungal collection (NRRL) in Peoria, IL (Accession #66828). Slow-growing 15
colonies that were slightly raised off the media with an undulated margin, typical of Sphaerulina 16
musiva, were observed on APDA in all cultures. Pycnidia formed on 7-week old cultures that 17
were incubated at 23 °C in the dark, and twenty conidia in 5% KOH were measured. Cylindrical 18
conidia measuring 18.2 (14.9-21.5) x 2.4 (2.0-3) µm on average had tapered apices and were 19
straight to slightly curved typical of S. musiva. DNA was extracted from mycelium, and PCR 20
amplification of the internal transcribed spacer (ITS) region was performed with ITS1F and ITS4 21
as previously described (White et al. 1990). Purified PCR products were sequenced, assembled 22
and visually edited, and deposited to GenBank (Accession # MH379707). The ITS sequence had 23
Page 1 of 3
Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-06-18-0928-PDN • posted 10/04/2018
This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ.
99% homology with deposited sequences labeled with the teleomorph name Mycosphaerella 24
populorum (GeneBank Accession #GU130032.1 and AY752863.1) or with the anamorph name 25
Sphaerulina musiva (GeneBank Accession #KF251619.1). The difference in deposited ITS 26
sequences is likely due to intraspecific variation. Pathogenicity tests were conducted twice to 27
determine if our isolate of S. musiva was the causal agent of the cankers observed on Populus 28
purdomii. Five (experiment 1) and nine (experiment 2) 60 day old, disease-free rooted-cuttings 29
of P. purdomii were inoculated in a greenhouse on February 15, 2018 (experiment 1) and April 30
16, 2018 (experiment 2) by placing a colonized 7 mm agar plug of a S. musiva isolate or non-31
infested APDA agar plug (n=3) into a sapwood wound made with a sterile #3 cork borer that was 32
then wrapped in Parafilm. Rooted cuttings were planted in a soilless medium, and were hand-33
watered every other day for the duration of the experiment. Plants were scored for canker 34
development on April 16, 2018 (experiment 1) and May 30, 2018 (experiment 2). All inoculated 35
trees showed sunken cankers outside the point of inoculations in both experiments, while the 36
mock-inoculated controls showed no disease symptoms. Isolates of Sphaerulina musiva were 37
successfully reisolated from all inoculated saplings, fulfilling Koch’s postulates. Isolate identity 38
was validated with colony morphology and ITS sequencing. To our knowledge, this is the first 39
report of Sphaerulina musiva causing branch cankers on Populus purdomii in the United States. 40
Populus purdomii has potential for use in breeding programs aimed at developing fast growing 41
hybrids intended for biomass production. However, hybrids produced will require careful 42
selection for resistance to S. musiva, or trees will have to be planted in areas where this disease is 43
of low incidence. 44
45
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Page 2 of 3
Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-06-18-0928-PDN • posted 10/04/2018
This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ.
References 47
48
Bier, J. 1939. Septoria canker of introduced and native hybrid poplars. Canadian Journal of Research 49
17:195-204. 50
White, T. J., Bruns, T., Lee, S., and Taylor, J. 1990. Amplification and direct sequencing of fungal 51
ribosomal RNA genes for phylogenetics. PCR Protocols: a guide to methods and applications 52
18:315-322. 53
54
Page 3 of 3
Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-06-18-0928-PDN • posted 10/04/2018
This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ.
