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First Report of Leaf Spots Caused by Alternaria alternata on Aquilegia flabellata in Italy
A. Garibaldi, D. Bertetti, G. Tabone, and M. L. Gullino
Fan columbine (Aquilegia flabellata), in the Ranunculaceae family, is used both in mixed
borders for gardening and for cut flower production. During spring 2021, 3-month-old potted
plants of A. flabellata growing in a glasshouse of the Agroinnova Centre (Grugliasco, Torino
province, Northern Italy), showed irregular, brown discolorations spread over a large part of
the leaf surface and petioles and were followed by necrosis up to 5.0 mm in size. The most
affected leaves turned yellow and dried. The disease incidence was 22% of 100 plants. Twenty
symptomatic leaves were immersed in 1% sodium hypochlorite for 30 s, then washed in sterile
water. Three fragments per leaf (about 1 mm2) were excised from the margins of the necrosis
and were plated on potato dextrose agar (PDA), obtaining 50 dark green fungal colonies. Five
pure colonies obtained by hyphal tip transfers were grown on potato carrot agar (PCA;
Simmons 2007), under a light/dark regime (14/10 h). They shared similar morphological
characteristics, producing conidiophores with branching chains of brown, septate, ovoid to
obclavate conidia. Conidia had one to four transverse and zero to two longitudinal/oblique
septa and measured 8 to 28 × 6 to 15 (average 19 × 9) μm (n = 50). Sometimes conidia had a
beak that was 2 to 10 (mean 4) μm long. These morphological features led to classifying the
fungus as Alternaria sect. Alternaria (Woudenberg et al. 2015). The DNA of a representative
isolate (21/59-9) was extracted. PCR was performed for the nuclear ribosomal internal
transcriber spacer (ITS) region (White et al. 1990), translation elongation factor 1 alpha (tef1),
and endopolygalacturonase (endoPG) gene regions (Woudenberg et al. 2015) (GenBank accession
nos. MZ474198, OL990028, and OL990029, respectively). Sequences had 100% (478/478 bp)
similarity for the ITS region with the ex-type CBS 130265 of Alternaria alternata (GenBank
Accession No. KP124391) and 100% (240/240 bp) and 99.55% (446/448 bp) similarity for tef and
endoPG (GenBank accession nos. KP125105 and KP124031, respectively) with the ex-type CBS
102598 of A. alternata. In the pathogenicity test, a conidial suspension (1.0 × 105 conidia/ml)
was prepared from the isolate 21/59-8 and sprayed on basal nonwounded leaves and petioles
of three 7-month-old healthy plants of A. flabellata. Three plants of the same host were
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A. Garibaldi, D. Bertetti, G. Tabone, and M. L. Gullino
Fan columbine (Aquilegia flabellata), in the Ranunculaceae family, is used both in mixed
borders for gardening and for cut flower production. During spring 2021, 3-month-old potted
plants of A. flabellata growing in a glasshouse of the Agroinnova Centre (Grugliasco, Torino
province, Northern Italy), showed irregular, brown discolorations spread over a large part of
the leaf surface and petioles and were followed by necrosis up to 5.0 mm in size. The most
affected leaves turned yellow and dried. The disease incidence was 22% of 100 plants. Twenty
symptomatic leaves were immersed in 1% sodium hypochlorite for 30 s, then washed in sterile
water. Three fragments per leaf (about 1 mm2) were excised from the margins of the necrosis
and were plated on potato dextrose agar (PDA), obtaining 50 dark green fungal colonies. Five
pure colonies obtained by hyphal tip transfers were grown on potato carrot agar (PCA;
Simmons 2007), under a light/dark regime (14/10 h). They shared similar morphological
characteristics, producing conidiophores with branching chains of brown, septate, ovoid to
obclavate conidia. Conidia had one to four transverse and zero to two longitudinal/oblique
septa and measured 8 to 28 × 6 to 15 (average 19 × 9) μm (n = 50). Sometimes conidia had a
beak that was 2 to 10 (mean 4) μm long. These morphological features led to classifying the
fungus as Alternaria sect. Alternaria (Woudenberg et al. 2015). The DNA of a representative
isolate (21/59-9) was extracted. PCR was performed for the nuclear ribosomal internal
transcriber spacer (ITS) region (White et al. 1990), translation elongation factor 1 alpha (tef1),
and endopolygalacturonase (endoPG) gene regions (Woudenberg et al. 2015) (GenBank accession
nos. MZ474198, OL990028, and OL990029, respectively). Sequences had 100% (478/478 bp)
similarity for the ITS region with the ex-type CBS 130265 of Alternaria alternata (GenBank
Accession No. KP124391) and 100% (240/240 bp) and 99.55% (446/448 bp) similarity for tef and
endoPG (GenBank accession nos. KP125105 and KP124031, respectively) with the ex-type CBS
102598 of A. alternata. In the pathogenicity test, a conidial suspension (1.0 × 105 conidia/ml)
was prepared from the isolate 21/59-8 and sprayed on basal nonwounded leaves and petioles
of three 7-month-old healthy plants of A. flabellata. Three plants of the same host were
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