Ewa Mirzwa-Mróz

First report of Microcyclosporella mali causing sooty blotch and flyspeck disease on plum in Poland.

Sooty blotch and flyspeck (SBFS), a disease caused by a complex of fungi, results in substantial economic losses for commercial growers of scab-resistant apple (Malus × domestica Borkh.) cultivars in Poland. However, many species causing SBFS in Poland are unidentified and sources of inoculum are uncertain. In August 2009, signs of SBFS were noted on fruit of plum (Prunus domestica L., cvs. Sweet Common Prune and Oullins Golden Gage) in orchards near Mostki in central Poland. Colonies consisted of olive green-to-black mycelial mats with few sclerotium-like bodies; infections ranged in severity from scattered spots to nearly complete coverage of the fruit surface. Ten of these colonies were isolated on potato dextrose agar (PDA). After 10 days of incubation at 22°C, total DNA was extracted; amplification of the internal transcribed spacer (ITS) region of rDNA utilized primers ITS1 and ITS4 (1). Nucleotide sequences were analyzed by ClustalW and compared with sequences in GenBank using BLAST. Sequences showed 99 to 100% homology to Microcyclosporella mali (2), which was formerly assigned as Pseudocercosporella sp. (1). Sequences from five isolates were submitted to GenBank (Accession Nos. HM101275, HM101276, HM101277, HM101278, and HM101279). Morphological characteristics-conidiogenous cells integrated, sympodial and polyblastic; conidial scars nonthickened and inconspicuous; conidia hyaline, subcylindric, narrow, straight or very slightly curved, truncate at the base and obtuse at the apex, often catenulate in simple or branched chains, with one (commonly) to five septa (12.5 × 2.6 to 50.7 × 4.0 μm)-were consistent with descriptions of M. mali (2). To fulfill Kochs postulates, each of the 10 isolates was used to inoculate three healthy apple fruit (cv. Golden Delicious) that had been previously washed under tap water and disinfested with 70% ethanol. After fruit were swabbed with cotton plugs that had been saturated with a suspension of spores in sterile distilled water (SDW), inoculated fruit were placed on filter paper that had been moistened with SDW, then sealed in foil bags and incubated at 22°C. When bags were removed 5 weeks later, dark colonies had appeared on the fruit. Isolates obtained from these colonies were morphologically identical to those used for inoculation. Control (SDW-inoculated and noninoculated) fruit that were incubated in the same manner developed no colonies. To our knowledge, this is the first report of SBFS on plum caused by M. mali in Poland; it had previously been noted as part of the SBFS complex on apple in Germany and Slovenia (2) and on apple and plum in the United States (3). References: (1) J. C. Batzer et al. Mycologia 97:1268, 2005. (2) J. Frank et al. Persoonia 24:93, 2010. (3) J. Latinović et al. Plant Dis. 91:1685, 2007.

First Report of Alternaria Black Spot Caused by Alternaria alternata on Blue Honeysuckle in Poland

Blue honeysuckle (Lonicera caerulea L. var. kamtschatica Sevast.) is becoming increasingly popular in Poland. Each year, the growing area of this plant increases significantly because it produces one of the earliest crops, and its berries contain a high concentration of antioxidants (i.e., vitamins A and C, potassium, calcium, phenolic compounds, and anthocyanins). Symptoms of Alternaria leaf spot were observed on blue honeysuckle plants growing in allotment gardens in Kanie (52°08′08″N, 20°47′14″E, Mazovia Province) and Kopalino (54°47′34″N, 17°50′02″E, Pomerania Province) in 2015 to 2016. The initial infections were noted on leaves at the end of June. Under favorable conditions, complete defoliation was observed at the beginning of August. Symptoms on leaves consisted of oval to irregularly shaped spots, 1 to 4 mm in diameter. They were initially black and later turned brown with a dark brown to black border. Over time, the center of the spots developed a gray shade. The spots were surrounded by a yellowish halo. The lesions gradually enlarged and sometimes coalesced. In 2015 and 2016, infected leaves were collected and sections (3 × 4 mm) were excised from the margin of diseased leaf tissue, disinfested with 1% sodium hypochlorite for 2 min, transferred to potato dextrose agar (PDA), and incubated at 25 ± 2°C. Twenty single-spored isolates were obtained from the developed colonies of the fungus. On potato carrot agar (PCA), cultures were olive brown with sparse, white-gray aerial mycelium. The conidiospores were obpyriform to obclavate, light brown to dark brown, with 1 to 6 transepta and 0 to 2 longisepta. After 9 days on PCA, 100 conidia were measured to be 12 to 46 × 7 to 17 µm. Morphological characteristics were similar to those described for Alternaria alternata (Fr.) Keissl (Simmons 2007). For the pathogenicity test, four isolates were grown on salt nutrient agar (SNA) for 10 days. Inoculations were performed on 2-year-old plants of blue honeysuckle cultivars Wojtek and Duet growing in pots. Plant leaves (three plants per isolate) were rinsed with sterile water. Next, they were inoculated with a spore suspension (∼10⁶ spores/ml) of each isolate using a pipette. Control plants were treated with sterile water. All plants were incubated in a moist chamber (Sanyo) at 27°C during the day and 20°C during the night, 90% relative humidity, and 16-h photoperiod. After 10 days, first leaf spots developed on the inoculated plants. The control plants remained symptomless. The pathogen was successfully reisolated from the spots and confirmed to be identical with the original fungal isolate, fulfilling Koch’s postulate. To confirm the morphological identification, genomic DNA was extracted from four single-spore isolates (JK8, JK2.3, JK 4.2, and Alt4). The internal transcribed spacer (ITS1, 5.8S, and ITS2) region of rDNA and the translation elongation factor (TEF1-α) gene were amplified with primers ITS1 and ITS4 (White et. al 1990) and EF1-728F (Carbone and Kohn 1999) and EF2 (O’Donnell et al. 1998), respectively. One representative sequence for the ITS and one for the TEF1-α gene were deposited in GenBank (accession nos. MF564200 and MF564197), respectively. BLAST analysis showed 99% identity with A. alternata sequences published in GenBank at the ITS (AF347031) and TEF1-α (KC584634) regions. Based on the morphology and molecular studies, the causal agent of Alternaria black spot on the blue honeysuckle was identified as A. alternata. To our knowledge, this is the first report of this pathogen on blue honeysuckle worldwide.

Valdensinia heterodoxa Peyronel as a New Pathogen of Blueberry in Poland

Valdensia leaf blight on blueberry in Poland was reported in one commercial nursery plantation near Prażmów, Mazovia voivodship, where heavy defoliation was observed on cv. Bluecrop, grown in nursery pots, in August 2011. Older fruiting bushes were only slightly affected by the disease. Initial symptoms of the disease were small, oval to circular zonated necrosis surrounded with dark brown borders that enlarged on the leaves throughout the canopy. Multicellular, hyaline or light brown, star-shaped conidiospores were observed on the necrotic areas. The mean length of 50 conidiospores from the end of head to the end of arm apex was 307 to 348 μm (4). Eight single-spore isolates of the fungus were obtained. Single conidiospores were picked up from necrotic spots on leaves and transferred with sterile needle on potato dextrose agar (PDA) and incubated at 20°C under ambient light. After 10 days of incubation, total DNA was extracted. Amplification of the internal transcribed spacer (ITS) region of rDNA was done using primers ITS1F and ITS4A (1). PCRs were carried out as follows: initial denaturation at 94°C for 2 min, denaturation at 94°C for 1 min, annealing at 57°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 5 min for 28 cycles (Applied Biosystems Veriti 96 Wel Thermal Cycler). Amplicons, which were approximately 520 bp, were sequenced and nucleotide sequences were analyzed by Clustal W2EBI. The sequences of all eight isolates showed 100% similarity to each other and were compared with sequences stored in GenBank using BLAST. Sequences were 525 bp long and showed 100% homology to Valdensinia heterodoxa Peyronel, Sclerotiniaceae (anamorph: Valdensia heterodoxa Peyronel) from Japan and Norway (Accession Nos. AB663682 and Z81447, respectively) (3). The sequence from one isolate was submitted to GenBank (Accession No. KF212190). To fulfill Kochs postulates, each of the eight isolates was used to inoculate 20 healthy young leaves of Vaccinium corymbosum L. cv. Bluecrop and bilberry (V. myrtillus L.) (10 leaves per plant). Mycelial plugs 5 mm in diameter were taken from PDA cultures, approximately 20 days old, and used as inoculum and placed in the center of each leaf and moistened with sterile distilled water. Mycelium-free plugs were used as control. Inoculated leaves were placed in plastic box and incubated at 20°C in laboratory for 5 days, at which time small necrotic lesions consistent with initial symptoms of the disease were observed. Isolates obtained from these symptoms were morphologically identical to those used for inoculation. Control leaves did not show any disease symptoms. Valdensia leaf blight occurrence may be attributed to rainy July and August 2011 and long presence of water on soil surface. In Poland, Valdensinia heterodoxa causes heavy defoliation of Vaccinium myrtillus in pine stands and is a common pathogen of some herbaceous plants (2). To our knowledge, this is the first report of Valdensia leaf blight on highbush blueberry in Poland. References: (1) I. Larena et al. 75:187, 1999. (2) W. Mułenko and S. Woodward. Mycologist 10:69, 1996. (3) S. Nekoduka et al. J. Gen. Plant Pathol. 78:151, 2012. (4) S. Zhao and S. F. Shamoun. Mycology 1:113, 2010.