Brown rot caused by Monilinia fructicola is one of the most important diseases of peach. The pathogen is included in the EPPO A2 list of quarantine organisms for Europe (2). M. laxa and M. fructigena are common in Hungary, but M. fructicola has never been reported in orchards, in trade, or in markets. In early October 2005, brown rot was observed on imported peaches from Italy and Spain at a vegetable market and some supermarkets in Budapest. The variety of peach was identified as ‘Michellini’ by colleagues in the Department of Pomology of Corvinus University. The pathogen was identified as M. fructicola on the basis of morphological and molecular characteristics. Symptoms began with a small, circular brown spot, and the rot spread rapidly. At the same time, numerous small, grayish stromata developed. Finally, the whole surface of the fruit was covered with conidial tufts. The conidia were one-celled, lemon-shaped, hyaline, 15.7 × 10.3 μm, and produced in branched monilioid chains. Conidia from infected fruit were transferred to potato dextrose agar. Fungal mycelium grew at a linear rate of 10.7 mm per 24 h at 22°C in the dark. The color of the colony was grayish, and the sporulation showing concentric rings was abundant (sporulation is sparse in M. laxa or M. fructigena). The colony was not rosetted and the margin was not lobed, in contrast with M. laxa. Pathogenicity was tested by inoculating surface-sterilized, mature peach fruits with conidia. Inoculated and control fruits were placed in a sterilized glass container at room temperature. After 5 days of incubation, typical brown rot symptoms developed on inoculated fruits while control fruits remained healthy. M. fructicola was reisolated from the inoculated fruits. PCR was used to identify the fungus (1). Species-specific internal transcribed spacer (ITS) primers for M. fructicola, M. laxa, and M. fructigena were used to amplify the DNA of isolates. Three type-cultures were used as the positive control. Following the removal of the mycelia from the agar, total DNA was extracted using a cetyltrimethylammoniumbromide extraction. The nucleic acid-containing pellet was resuspended in RNase containing Tris-EDTA buffer. DNA quality was assessed by gel electrophoresis on 1% agarose gel stained with ethidium bromide. The molecular genetic identification method confirmed the results of morphological identification. To our knowledge, this is the first report of M. fructicola on peaches in Hungary or in eastern Europe. References: (1) R. Ioos and P. Frey. Eur. J. Plant Pathol. 106:373, 2000. (2) OEPP/EPPO. List of A2pests regulated as quarantine pests in the EPPO region. Version 2005-09. Online publication, 2005.
A Monilinia fructigena-like isolate (UFT) was collected from apple shoots in northeastern Hungary (Újfehértó). Brownish dieback and buffcoloured stromata were observed on shoots and small fruits of cv. 'Ashton Bitter'. On potato dextrose agar (PDA) the colonies were yellowish in colour and irregular black stromatal crusts occurred. Conidia (16.6 × 10.1 µm) were slightly smaller than the average of M. fructigena. The fungus caused brown rot on inoculated apple fruits, and produced numerous sporodochia. The sequences of the rDNA internal transcribed spacer regions of the UFT isolate were almost identical to that of a previously described Monilia polystroma isolate, containing all five nucleotides that distinguish it from M. fructigena. Comparison of a genomic sequence of unknown function revealed that repetitive sequence motifs occurred in different numbers as insertions in the genomes of M. fructigena, Monilia polystroma, and the UFT isolate. Classical and molecular characterisation indicated that the UFT isolate belonged to Monilia polystroma. To our knowledge this is the first report of Monilia polystroma in Europe.
Monilinia is a well-known pathogen of fruit trees affecting fruit production all over the world. Three species of the Monilinia genus are particularly important with regard to fruit trees and ornamentals, causing serious blossom and twig blight and brown rot in fruits: Monilinia fructicola, Monilinia fructigena, and Monilinia laxa. In this study, Monilinia isolates were compared and identified using classical and molecular methods. Morphological and culture characteristics were determined and pathogenicity testing performed. In addition, internal transcribed spacer regions and a genomic sequence with unknown function were analyzed and compared with sequence data from other Monilinia species in an international database. Four Monilinia/Monilia species were identified: M. fructicola, Monilia polystroma, M. fructigena, and M. laxa. M. fructicola was isolated from imported peach fruits. M. polystroma was first reported from Hungary and Europe on apple shoots and fruits. M. fructigena was identified on tea-rose hybrid pseudofruits, which is the first occurrence of this pathogen on this host. M. laxa causes brown rot of grapes, which has only been reported in New Zealand. Substitutions and insertions were detected when comparing M. laxa, M. fructigena, and M. polystroma sequences. In the genomic sequence with unknown function, three repetitive sequence motifs were identified in different numbers, depending on species and isolate. On the phylogram produced in this analysis, the Hungarian M. polystroma isolate (UFT) and M. polystroma reference isolates localized at a different branch than the closely related M. fructigena isolates and other Monilinia species.
Pomegranate (Punica granatum L.), the hystoric fruit and ornamental crop native to Iran and North India is widely planted in the Mediterranean and became popular in the house gardens of northest parts of Europe (Fernandez et al. 2014) including Hungary. In August 2020 necrotic black lesions and serious defoliation were observed on 60% of 1-3 year old pomegranate trees (cv. Wonderful) in a horticultural nursery near Gödöllő, Hungary (47°36'00.9"N 19°21'26.5"E). Symptoms started as small irregular dark brown spots on the leaves, which later increased in size (2.6 ± 0.9 mm). Ultimately, the entire leaf turned yellow, defoliation resulted in damage on (6) – 8 – (15)% of the leaves. Then, black pycnidia with unicelled, elliptical to fusiform, colourless conidia (Avg. 50 conidia: 2.4 – (3.6) – 3.9 × 10.2 – (13,1) – 17.9 µm) developed on the surface. These morphological features matched those described earlier by Van Niekerk et al. (2004) and Alvarez et al. (2016) for C. granati. Conidia from pycnidia were directly transferred to potato dextrose agar (PDA) by sterile needle. The plates were incubated at 24°C in the dark. Light yellow colonies with whitish aerial mycelia and later black globose pycnidia were observed. Mass of conidia oozed from pycnidia after 15 days of incubation. Pathogenicity tests were carried out on 1-year-old potted P. granatum trees (cv. Wonderful) with 5 replicates in the greenhouse. Ten, randomly selected leaves were inoculated per plant. 7-mm mycelial plugs from the edge of 10-day-old colonies were placed directly on disinfested (2% NaOCl solution, than sterile distilled water) leaves. The plants were covered with plastic film for 3 days after inoculation (26±3°C and 87±3% relative humidity). Pathogenicity was also tested on nonwounded, surface-disinfested fruits by mycelial plugs in 3 × 3 replicates. Inoculated fruits were placed in large grass vessels for 15 days (24±2°C and 80±5% relative humidity). Uncolonized, sterile PDA plugs were used as controls in both cases. Dark brown legions developed after 9-12 days on the plants in the greenhouse. On pomegranate fruits, the fungus colonized the fruit after 7-8 days, followed by fruit rot. In some cases, after 2 weeks pycnidia developed on the skin surface. No decay were present on control leaves or fruits. The pathogen was reisolated from all infected tissues and identified as C. granati, thus fulfilling Koch's postulates. For molecular identification, total genomic DNA of the isolate was extracted from the growing margins of colonies on PDA and partial sequence of internal transcribed spacer (ITS) and translation elongation factor 1-alpha (tef1) were amplified by PCR using primers described by Alvarez et al. (2016). Sequence data of the Hungarian isolate of the ITS region (GenBank acc. no. MW581953) showed 99.8% identity (559 bp out of 560 bp) with C. granati sequences deposited in GeneBank (Acc. nos. MH860368, MH855389 and KX833582). Considering tef1 sequence of the Hungarian isolate (OM908764) obtained had complete identity with other published C. granati isolates (KX833676, KX833682). C. granati has been previously reported on pomegranate from Europe (Palou et al. 2010, Pollastro et al. 2016). Based on morphological and molecular studies, this is the first record of C. granati in Hungary. The economic importance of this disease in currently limited in Hungary due to pomegranate is rather an ornamental crop, however, the first cultivation trials have been already started. There is a risk that the spread of the pathogen began with the infected propagating material, as a result the disease may outbreak anywhere in the country.
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