J Gen Plant Pathol (2016) 82:174–176 DOI 10.1007/s10327-016-0649-8

DISEASE NOTE

Anthracnose of black locust caused by Colletotrichum nymphaeae (Passerini) Aa Naho Yamagishi1,3 • Toyozo Sato2 • Izumi Chuma1 • Yoshiyuki Ishiyama3 Yukio Tosa1

•

Received: 3 September 2015 / Accepted: 25 December 2015 / Published online: 21 March 2016 Ó The Phytopathological Society of Japan and Springer Japan 2016

Abstract Brown spots were found on leaves of black locust (Robinia pseudoacacia L.) in Nagano Prefecture, Japan, in August 2011. A fungus isolated from the diseased leaves reproduced the symptoms after inoculation, and was re-isolated from the lesions. The pathogen was identified as Colletotrichum nymphaeae based on morphological and molecular analyses. This is the first report of C. nymphaeae as a pathogen of anthracnose of black locust in Japan. Keywords Anthracnose  Black locust  Colletotrichum nymphaeae  Robinia pseudoacacia Black locust (Robinia pseudoacacia L.; Fabaceae), a tall tree native to North America, is recognized as an ‘‘alien species that is important in industry and requires appropriate management’’ (Ministry of the Environment of Japan 2015). In August 2011, small brown spots were found on leaves of black locust in a forest near a field in the central area of Nagano Prefecture. The lesions gradually enlarged to rounded brown spots (Fig. 1a). Small brown spots also appeared on petioles. These developed into sunken and fusiform spots (Fig. 1b).

& Naho Yamagishi [email protected] 1

Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan

2

Genetic Resources Center, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki 305-8602, Japan

3

Nagano Vegetable and Ornamental Crops Experiment Station, 1066-1 Soga, Tokoo, Shiojiri, Nagano 399-6461, Japan

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For isolating the causal agent, small pieces of leaf lesions were soaked in 70 % (v/v) ethanol for 30 s, washed in sterilized water, air-dried, and incubated on 2 % water agar containing 250 ppm chloramphenicol at 25 °C for 7 days. Conidia produced on the agar were streaked onto potato dextrose agar (PDA, Nissui Pharmaceutical, Tokyo, Japan) plates. Three conidia with agar pieces were picked from the plate using a microscope and transferred to separate PDA slants. These single-conidial isolates were designated as NACs1, NACs2, and NACs3, respectively. Pathogenicity of the isolates was checked by spraying a conidial suspension (1 9 105 conidia/mL) of isolate NACs1 was sprayed onto intact young commercial black locus plants (cultivar unknown), approximately 20 cm tall, purchased from a market. Three plants were covered with a transparent plastic bag for 1 day at 25 °C after inoculation. After removing the bag, the inoculated plants were cultivated for 10 days in a growth chamber at 25 °C with 12 h light/12 h dark. Three plants were sprayed with sterilized distilled water as controls. Ten days after inoculation, the symptoms were reproduced on the inoculated leaves and petioles (Fig. 1c, d); no symptoms were observed on the control. A fungus similar to isolate NACs1 was consistently reisolated from the reproduced lesions, but never from control leaves. Morphological characteritics of NACs1 were examined with a microscope after incubation on synthetic nutrientpoor agar (SNA) at 20 °C with 12 h light/12 h dark for 10 days. Its conidia were 1-celled, smooth, hyaline, tapered to the base, rounded at apex, 9.1–14.6 9 2.7–4.5 lm, with average L/B of 3.1 (Fig. 1e). Appressoria were aseptate, brown, ellipsoid, entire, 4.7–11.8 9 3.4–8.3 lm (Fig. 1f). Colonies on SNA were hyaline with low, aerial, white mycelium (Fig. 1g). These morphological characteristics were similar to those of Colletotrichum nymphaeae

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Fig. 1 Symptoms of black locust anthracnose and morphology of its causal agent, Colletotrichum nymphaeae. Brown spot on leaf (a) and sunken spot on petiole (b) of black locust collected in fields. Inoculated leaves (c) and petiole (d) of black locust, 10 days after

inoculation. Conidia (e) and appressoria (f) of isolate NACs1 formed on SNA after incubation at 20 °C for 10 days. Mycelial colony of the isolate NACs1 grown on SNA plate (g) or PDA plate (surface view: h, reverse view: i) at 20 °C for 10 days

Table 1 Morphological comparison of a pathogenic isolate responsible for black locust anthracnose with a previous description of Colletotrichum nymphaeae Isolate

Colony on PDA

Colony on SNA

Sclerotium

Conidium (on SNA)

Appressorium (on SNA)

Length 9 breadth (lm)

L/B ratio

Length 9 breadth (lm)

L/B ratio

NACs1

Gray, cottony, reverse pale gray to pale orange

Hyaline with low white aerial mycelium

Absent

9.1–14.6 9 2.7–4.5

3.1

4.7–11.8 9 3.4–8.3

1.5

C. nymphaeaea

ndb

Hyaline with low white aerial mycelium

Absent

(6-)9.5–13.5(-15) 9 (2-)3–4.5

3.1

(4-)5.511(-17) 9 (3-) 4-6.5(-9)

1.6

a

Damm et al. (2012)

b

Not described

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(Passerini) Aa (Damm et al. 2012) (Table 1). Colonies on PDA were gray and cottony and pale gray to pale orange in reverse (Fig. 1h, i). The isolate grew on PDA plates at 10–35 °C with maximum growth of 3.4 mm/day at 27 °C. The morphological and cultural characters of NACs1 agreed well with those of C. nymphaeae (Sato and Moriwaki 2013). For molecular support of these observations, genomic DNA of NACs1 was extracted from its mycelium grown on a PDA plate according to the method of Moriwaki et al. (2002). The rDNA-ITS region was amplified and sequenced using primer pair ITS5 and ITS4 (White et al. 1990) and the DNA as a template. The polymerase chain reaction was performed with conditions and procedures described in the previous report (Sato and Moriwaki 2013). Five hundred and nineteen base pairs of the rDNA-ITS sequence had 100 % identity with that of MAFF 242590 (AB618089), a strain of C. nymphaeae causing celery stunt anthracnose (Fujinaga et al. 2011; Yamagishi et al. 2015). Damm et al. (2012) reported that C. nymphaeae could be distinguished from other species on the basis of b-tubulin sequences. Therefore, we also analyzed the b-tubulin-2 gene of NACs1. Direct sequencing of the b-tubulin-2 gene of NACs1 as described by Sato and Moriwaki (2013) was unsuccessful for unknown reasons, so we analyzed this gene via cloning in a plasmid vector. A mycelial culture grown on SNA was harvested, suspended in 100 lL Tris– HCl (10 mM), and incubated at 95 °C for 10 min. The resulting lysate was used as a template. The b-tubulin-2 gene was amplified with primers T1 (50 -AACATGCGTGAGATTGTAAGT-30 ) (O’Donnell and Cigelnik 1997) and bt2 (50 -ACCCTCAGTGTAGTGACCCTTGGC-30 ) (Glass and Donaldson 1995) in a 50 lL reaction containing 1 U KOD FX Neo DNA polymerase (TOYOBO, Osaka, Japan), 1 9 PCR buffer for KOD FX Neo, 0.4 mM each of dNTPs, 0.3 lM primers, and 1 lL of the mycerial lysate, by PCR conditions, 2 min at 94 °C, and 30 cycles of 10 s at 98 °C, 30 s at 55 °C, and 1 min at 68 °C. A 780-bp amplicon was ligated with pBluescript II SK (?), to transform Escherichia. coli, and sequenced using M13-20 (50 -GTAAAACGACGGCCAGT-30 ) and M13 reverse (50 GGAAACAGCTATGACCATG-30 ) primers. Nucleotide sequences obtained were 100 % identical to AB618090, the C. nymphaeae b-tubulin-2 gene of strain MAFF 242590. Since the morphological characters matched the original description and the rDNA-ITS and b-tubulin-2 sequences have 100 % similarity with the reference strain, we identified NACs1 as C. nymphaeae. Colletotrichum destructivum O’Gara, Colletotrichum truncatum (Schweinitz) Andrus & W.D. Moore (syn.

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J Gen Plant Pathol (2016) 82:174–176

Colletotrichum glycines Hori), and Glomerella cingulata (Stoneman) Spaulding & H. Schrenk (syn. Guignardia robiniae Kaz. Ito & Tak. Kobayashi) were recorded as pathogens of black locust in Japan (Ito and Kobayashi 1958; PSJ [Phytopathological Society of Japan] and NIAS [National Institute of Agrobiological Sciences] 2012). This is the first report of anthracnose of black locust caused by C. nymphaeae. Therefore, we propose to add C. nymphaeae as a pathogen of this disease. Isolate NACs1 was deposited in NIAS Genebank, National Institute of Agrobiological Sciences (accession MAFF243458). Nucleotide sequence data of rDNA-ITS and b-tubulin-2 are available in the DDBJ/EMBL/GenBank databases under accession numbers LC091037 and LC089866, respectively.

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