Detection and Identification of Six Monilinia spp. Causing Brown Rot Using TaqMan Real-Time PCR from Pure Cultures and Infected Apple Fruit

Brown rot is a severe disease affecting stone and pome fruit. This disease was recently confirmed to be caused by the following six closely related species: Monilinia fructicola, M. laxa, M. fructigena, Monilia polystroma, M. mumecola, and M. yunnanensis. Because of differences in geographic distributions, some of these species are important quarantine pathogens in certain countries. In this study, we developed TaqMan real-time polymerase chain reaction (PCR) assays to detect and identify the six species. Primer pairs and probes were designed for Monilinia fructicola, M. fructigena, M. laxa, and Monilia polystroma based on sequence differences in the laccase-2 genes. Additionally, based on sequence differences in the elongation factor genes, primer pairs and probes were designed for Monilia mumecola and M. yunnanensis. The real-time PCR assays were able to specifically identify the target pathogens, with detection limits of 10 to 100 fg of DNA, which is equivalent to one to seven conidia. The assays were also able to detect the target pathogens in a mixed DNA sample comprising all six Monilinia spp. and related species. The real-time PCR assays accurately detected target fungi from infected apple fruit. Furthermore, the identification results were consistent with those of traditional morphological methods.

Suitability of different primers for specific molecular detection of Monilinia spp.

Monilinia spp. are economically important pathogens of pome and stone fruits. Four Monilinia species are present in Serbia - Monilinia fructigena, M. laxa, M. fructicola and Monilia polystroma. As detection and identification of Monilinia species are complex, the aim of this research was to evaluate species-specific primers in PCR in order to standardize fast and reliable molecular methods for differentiation between the four Monilinia species. Isolates of M. fructigena, M. laxa, M. fructicola and M. polystroma from apple fruit and referent isolates from Italy and Japan were used for testing. Specific molecular detection of M. laxa was obtained using ITS1Mlx/ITS4Mlx and Ml-Mfg-F2/Ml-Mfc-R1 primer pairs, and M. fructicola using ITS1Mfcl/ITS4Mfcl and Mfc-F1/Mfc-R1 primer pairs. ITS1Mfgn/ITS4Mfgn and ITS1/Mfg-R2 primer pairs, described as M. fructigena species-specific, amplified M. fructigena and M. polystroma, as well. Specific detection of these two species as well as of all four tested Monilinia species was obtained using the reverse primer MO368-5 with forward primers MO368-8R, Laxa-R2 and MO368-10R in separate or in Multiplex PCR reactions.

Monilinia Species Associated with Brown Rot of Cultivated Apple and Pear Fruit in China

Monilinia isolates were collected from major apple and pear production regions in China from 2004 to 2011 and identified based on their morphological characteristics and three highly conserved loci. The 247 isolates belonged to three species: Monilinia fructicola, Monilia yunnanensis, and Monilia polystroma. M. yunnanensis was the most prevalent (77%), followed by M. polystroma (20%) and Monilinia fructicola (3%). Monilia yunnanensis is primarily distributed in the south, north, and west of China; M. polystroma is limited to the north and east; and Monilinia fructicola was detected only from a few samples from the north and east. Phylogenetic analysis based on internal transcribed spacer, β-tubulin, and laccase (lcc2) genes suggested that Monilia yunnanensis, M. polystroma, and Monilinia fructigena are closely related, and Monilia yunnanensis is more distantly related. We also found that these three species do not show consistent differences in morphological characteristics, including colony morphology, colony expansion rate, conidial characteristics, and the amount of stroma produced in culture. Thus, these three species are more like phylogenetic species in the process of speciation. In addition, a set of species-specific primers based on single-nucleotide polymorphisms and deletions in the lcc2 gene region were designed and a conventional polymerase chain reaction method successfully developed for differentiating Monilinia fructicola, Monilia yunnanensis, M. polystroma, and Monilinia laxa from the other species.

First Report of Brown Rot Caused by Monilia polystroma on Apple in Serbia

Monilia polystroma van Leeuwen is a new Japanese species, similar to M. fructigena but distinguishable based on morphological and molecular characteristics (3). After its first discovery on apple in Japan, occurance of M. polystroma in Europe has been reported in Hungary, the Czech Republic, and Switzerland (2,3,4). In October 2011, during a survey for apple fungal pathogens in the Bela Crkva district, 15 apple fruit (Malus domestica Borkh.) cv. Golden Delicious were collected. Two isolates of Monilinia polystroma were obtained from apple fruit showing brown rot, covered with small yellowish sporodohia. The pathogen was identified as M. polystroma based on morphological and molecular features (1,3). Upon isolation, colonies cultivated on PDA were white to grayish and the mycelium grew 8.85 mm per day at 22 ± 1°C in 12-h light/12-h dark regime. After 6 to 8 days of incubation, black stromatal plates were observed on the reverse sides of the inoculated petri dishes. Conidia were one-celled, limoniform, hyaline, 14.7 to 21.88 μm (16.2 mean) × 7.85 to 12.92 μm (10.8 mean), and were produced in branched monilioid chains on inoculated apple fruit. Morphological identification was confirmed by PCR (1) using genomic DNA extracted from the mycelium of pure cultures, and amplified products of 425 bp in length, specific for M. polystroma were amplified as expected with primers MO368-5 and MO368-8R. For one isolate, the ribosomal ITS1-5.8S-ITS2 region was obtained, using primers ITS1 and ITS4, and deposited in GenBank (Accession No JX315717). The sequence was 498 bp in length and showed 100% identity with sequences deposited for M. polystroma in NCBI GenBank (JN128835, AM937114, GU067539). Pathogenicity was confirmed by wound-inoculating five surface-sterilized, mature apple fruit with mycelium plugs (5 mm in diameter) of both isolates grown on PDA. Control fruit were inoculated with sterile PDA plugs. After 3 days of incubation in plastic containers, under high humidity (RH 90 to 95%) at 22 ± 1°C, typical symptoms of brown rot developed on inoculated fruit, while control fruit remained symptomless. Isolates recovered from symptomatic fruit showed the same morphological and molecular characteristics as original isolates. To the best of our knowledge, this is the first report of M. polystroma in Serbia. Further studies are necessary to estimate the economic importance and geographic distribution of this organism in Serbia. References: (1) M.-J. Côté et al. Plant Dis. 88:1219, 2004. (2) M. Hilber-Bodmer et al. Plant Dis. 96: 146, 2012. (3) G. C. M. van Leeuwen et al. Mycol. Res. 106: 444, 2002. (4) OEPP/EPPO Reporting Service. Retrieved from http://archives.eppo.int/EPPOReporting/2011/Rse-1106.pdf

Population Structure of Brown Rot Fungi on Stone Fruits in China

In total, 455 Monilinia isolates from stone fruits collected from several provinces (cities) in China from 2003 to 2009 were identified to species based on morphological characteristics, molecular identification, and the sequence of the internal transcribed spacer (ITS) regions 1 and 2 and the 5.8S gene of the ribosomal RNA. Overall, four species were detected (Monilinia fructicola, M. fructigena, M. laxa, and Monilia polystroma). M. fructicola was the most prevalent (93.0%) followed by M. fructigena (4.8%), M. laxa (2.0%), and Monilia polystroma (0.2%). M. fructicola and M. fructigena were found on peach, plum, and apricot; M. laxa was found only on apricot, cherry (in an organic orchard), and wild peach; and Monilia polystroma was found only on plum in Heilongjiang. The pathogenicity of Monilinia fructicola, M. laxa, and M. fructigena did not significantly differ on wounded nectarine and apricot, indicating that the differences in frequency of occurrence were not linked to virulence. Phylogenetic analysis based on ITS sequences showed that the isolates of M. laxa and M. fructigena from China differed from isolates of these species from other countries, and that the difference led to the separation of the isolates from China and those from other countries into different phylogenetic groups. Further study is needed to determine whether they are cryptic species.

Monilia polystroma. [Distribution map].

A new distribution map is provided for Monilia polystroma G. Leeuwen. Ascomycota: Helotiales. Hosts: apple (Malus spp.), quince (Cydonia spp.), stone fruit (Prunus spp.) and pear (Pyrus spp.). Information is given on the geographical distribution in Europe (Hungary), Asia (China, Heilongjiang, Japan, Honshu).

First Report of Brown Rot on Plum Caused by Monilia polystroma in China

In August 2008, mummies of dwarf sweet plum (Prunus aitianli) fruit covered with grayish, conidial tufts were found in an orchard in Mudanjiang City of Heilongjiang in China. Conidial masses were touched with a sterilized wire loop and streaked onto the surface of water agar (WA) plates. After incubating at 22 ± 2°C for 16 to 24 h, individual germinated spores were picked out with a sterilized scalpel blade under a microscope in a laminar flow cabinet, and transferred to potato dextrose agar (PDA) in petri dishes. Mycelium grew an average of 10.7 mm per day on PDA and formed a white-to-grayish colony with irregular, black stroma 12 days after incubation at 22 ± 2°C under 12-h light/12-h dark. The average size of stroma was 8.19 cm2 per petri dish 37 days after incubation in the dark. The conidia were one-celled, hyaline, lemon-shaped, 15.2 (10.8 to 18.9) × 10.9 (8.3 to 16.3) μm, and arranged in branched monilioid chains on inoculated apples. The PCR products of internal transcribed spacer (ITS) region 1 and 2 and 5.8S gene of the ribosomal RNA amplified with primers ITS1 and ITS4 was directly sequenced in both directions using the PCR primers. The sequence of the Monilia polystroma isolate (GenBank Accession No. GU067539) was identical to the reference isolate of M. polystroma (CBS102686), containing five nucleotides that distinguish it from Monilinia fructigena (1,3). The pathogen was identified as M. polystroma on the basis of morphological characteristics (3) and the sequence of internal transcribed spacer (ITS) region 1 and 2 and 5.8S gene of the ribosomal RNA. Pathogenicity was confirmed by inoculating surface-sterilized, mature plum and apple fruit wounded with a nail, with a mycelial plug (5 mm in diameter) of the fungus at each wound. Fruit treated with plain PDA plugs were used as a control. Inoculated fruits were placed in a sterilized moist chamber at room temperature (23 to 28°C). Fifteen plums and nine apples were used in each of two replicated tests. All inoculated fruit developed typical brown rot symptoms 4 days after inoculation, while the control fruit remained healthy. M. polystroma was reisolated from the inoculated fruit and identified by the above methods. M. polystroma was first reported on apple in Japan (3) and it was recently discovered in an apple orchard in Hungary (2). Although the occurrence of Monilinia fructicola, Monilinia laxa, and Monilinia fructigena (teleomorphs of the three Monilia spp.) in China have been documented, to our knowledge, this is the first report of the occurrence of M. polystroma in China. References: (1) C. E. Fulton et al. Eur. J. Plant Pathol. 105:495, 1999. (2) M. Petróczy and L. Palkovics. Eur. J. Plant Pathol. 125:343, 2009. (3) G. C. M. van Leeuwen et al. Mycol. Res. 106:444, 2002.