Identification and mycotoxigenic capacity of fungi associated with pre- and postharvest fruit rots of pomegranates in Greece and Cyprus.

Pre- and postharvest fruit rots of fungal origin are an important burden for the pomegranate industry worldwide, affecting the produce both quantitatively and qualitatively. During 2013, local orchards were surveyed and 280 fungal isolates from Greece (GR) and Cyprus (CY) were collected from pomegranates exhibiting preharvest rot symptoms, and additional 153 isolates were collected postharvest from cold-stored fruit in GR. Molecular identification revealed that preharvest pomegranate fruit rots were caused predominately by species of the genera Aspergillus (Aspergillus niger and Aspergillus tubingensis) and Alternaria (Alternaria alternata, Alternaria tenuissima, and Alternaria arborescens). By contrast, postharvest fruit rots were caused mainly by Botrytis spp. and to a lesser extent by isolates of Pilidiella granati and Alternaria spp. Considering that a significant quota of the fungal species found in association with pomegranate fruit rots are known for their mycotoxigenic capacity in other crop systems, their mycotoxin potential was examined. Alternariol (AOH), alternariol monomethyl-ether (AME) and tentoxin (TEN) production was estimated among Alternaria isolates, whereas ochratoxin A (OTA) and fumonisin B2 (FB2) production was assessed within the black aspergilli identified. Overall in both countries, 89% of the Alternaria isolates produced AOH and AME in vitro, while TEN was produced only by 43.9%. In vivo production of AOH and AME was restricted to 54.2% and 31.6% of the GR and CY isolates, respectively, while none of the isolates produced TEN in vivo. Among black aspergilli 21.7% of the GR and 17.8% of the CY isolates produced OTA in vitro, while in vivo OTA was detected in 8.8% of the isolates from both countries. FB2 was present in vitro in 42.0% of the GR and 22.2% of the CY isolates, while in vivo the production was limited to 27.5% and 4.5% of the GR and the CY isolates, respectively. Our data imply that mycotoxigenic Alternaria and Aspergillus species not only constitute a significant subset of the fungal population associated with pomegranate fruit rots responsible for fruit deterioration, but also pose a potential health risk factor for consumers of pomegranate-based products.

Differences in Frequency of Transposable Elements Presence in Botrytis cinerea Populations from Several Hosts in Greece.

This study was conducted primarily to investigate the presence and frequency distribution of the transposable elements Boty and Flipper in populations of the necrotroph plant pathogen Botrytis cinerea in Greece. In total, 334 isolates were collected from diseased grape, strawberry, tomato, cucumber, kiwifruit, and apple fruit during 2009. The presence of the two transposable elements was based on polymerase chain reaction detection. Results showed that all the sampled hosts occurred in sympatry, with four possible different genotypes (transposa type carrying both transposable elements, Boty type carrying only the Boty element, Flipper type carrying only the Flipper element, and vacuma type carrying neither transposable element). Marked differences in genotype frequencies among populations were observed. In tomato, cucumber, grape, and strawberry, transposa isolates carrying both elements were predominant in the populations whereas, in kiwifruit and apple fruit populations, the vacuma isolates were prevailing. Furthermore, in kiwi and apple fruit populations, high frequencies of Flipper-type isolates were observed. In an attempt to explain the observed predominance of vacuma isolates in kiwifruit populations, the mycelial growth rate of a set of vacuma isolates was compared with the mycelial growth rate of a set of transposa isolates at three different temperatures (0, 10, and 20°C). The same set of isolates was used to compare pathogenicity of isolates on wound-inoculated kiwifruit incubated at two different temperatures (0 and 20°C), in terms of disease incidence and disease severity. In addition, the selected isolates were used to compare their ability in causing latent infections on kiwifruit in the field. The results showed that vacuma and transposa isolates had similar mycelial growth rates at the limiting temperatures of 0 and 10°C, while vacuma isolates grew faster at the optimum temperature of 20°C. Similarly, there was no significant difference regarding pathogenicity on kiwifruit between transposa and vacuma isolates. However, artificial inoculations conducted on blossoms in the field showed that vacuma isolates caused significantly higher incidence of latent infections.

Evaluation of the incidence of the G143A mutation and cytb intron presence in the cytochrome bc-1 gene conferring QoI resistance in Botrytis cinerea populations from several hosts.

BACKGROUND: Previous studies have shown that resistance of Botrytis cinerea to QoI fungicides has been attributed to the G143A mutation in the cytochrome b (cytb) gene, while, in a part of the fungal population, an intron has been detected at codon 143 of the gene, preventing QoI resistance. During 2005-2009, 304 grey mould isolates were collected from strawberry, tomato, grape, kiwifruit, cucumber and apple in Greece and screened for resistance to pyraclostrobin and for the presence of the cytb intron, using a novel real-time TaqMan PCR assay developed in the present study. RESULTS: QoI-resistant phenotypes existed only within the population collected from strawberries. All resistant isolates possessed the G143A mutation. Differences were observed in the genotypic structure of cytb. Individuals possessing the intron were found at high incidence in apple fruit and greenhouse-grown tomato and cucumber populations, whereas in the strawberry population the intron frequency was lower. Cultivation of QoI-resistant and QoI-sensitive isolates for ten culture cycles on artificial nutrient medium in the presence or absence of fungicide selection showed that QoI resistance was stable. CONCLUSIONS: The results of the study suggest that a high risk for selection of QoI-resistant strains exists in crops heavily treated with QoIs, in spite of the widespread occurrence of the cytb intron in B. cinerea populations. The developed real-time TaqMan PCR constitutes a powerful tool to streamline detection of the mutation by reducing pre- and post-amplification manipulations, and can be used for rapid screening and quantification of QoI resistance.