Postharvest Fruit Rots of Apple in Greece: Pathogen Incidence and Relationships Between Fruit Quality Parameters, Cultivar Susceptibility, and Patulin Production.

The incidence of pathogens associated with postharvest fruit rots on the four most extensively cultivated apple cultivars (Red Delicious, Golden Delicious, Granny Smith, and Fuji) in Greece was surveyed during two consecutive storage periods (2008-09 and 2009-10) in five packinghouses located in northern Greece. The fungi isolated were identified based on their morphological characteristics and internal transcribed spacer gene sequencing. In the four cultivars sampled, Penicillium expansum and Botrytis cinerea were the predominant pathogens, accounting for averages of 44.2 and 23.6%, respectively, of the pathogens isolated from the sampled fruit. Two other important rot pathogens were Alternaria tenuissima and Mucor pyriformis, accounting for 16.1 and 6.6%, respectively, of the diseased apple fruit. Other pathogens such as Monilinia laxa, M. fructigena, Botryosphaeria obtusa, Geotrichum candidum, Fusarium avenaceum, and F. proliferatum were isolated at low frequencies and are considered of minor importance. Measurements of the resistance level of the four apple cultivars to fruit rot caused by P. expansum and Botrytis cinerea revealed that Golden Delicious was the most susceptible to blue mold while Fuji was the most susceptible to gray mold infections. Susceptibility to gray mold was negatively correlated with flavonoid and phenol concentration as well to fruit antioxidant activity, while susceptibility to blue mold was negatively correlated with fruit firmness and phenol concentration. Patulin production was significantly higher in Red Delicious and Golden Delicious fruit than in Granny Smith and Fuji fruit and was negatively correlated with the acidity of the fruit. The high incidence of P. expansum and A. tenuissima along with the presence of F. avenaceum and F. proliferatum, all of which are potentially mycotoxin producers, emphasize the risk for mycotoxin contamination of apple fruit juices and by-products. Furthermore, information on the distribution of the pathogens on the main cultivars may be useful for the implementation of strategies to control the diseases and minimize the threat of mycotoxin contamination on each cultivar.

First Report of Botrytis cinerea Causing Gray Mold of Pomegranate (Punica granatum) in Greece.

Pomegranate is rapidly increasing in production in Greece. During August of 2008 in the region of Larisa (central Greece), preharvest fruit rot was observed on pomegranate (cv. Kapmaditika) that caused losses estimated at 10%. Symptoms first appeared as small spots on the fruits that later increased in size and developed into expanded, dark brown lesions. Internally, tissues were soft and brown with gray mycelia and conidiophores observed. Affected fruits decayed completely during 2 months of storage (5 to 6°C), causing yield losses of up to 20%. To isolate the casual agent, conidia and conidiophores were scraped aseptically from the internal tissues, suspended in sterile water, and streaked onto the surface of potato dextrose agar (PDA). Single hyphal tips were transferred to PDA, and the isolated fungus was identified as Botrytis cinerea Pers.:Fr. on the basis of morphological characteristics (2). B. cinerea was consistently isolated from symptomatic tissues. Colonies of B. cinerea on PDA were at first colorless and became gray to brown with the development of lemon-shaped conidia (average 7.5 × 9 µm). Sclerotia were black and varied in size (1.4 to 4.5 × 1.5 to 2.7 mm) and shape (2). Pathogenicity of the isolated fungus was tested by wound inoculating five mature pomegranate fruits (cv. Kampaditika) after surface sterilization with 5% sodium hypochlorite. Plugs of the fungus (5 mm in diameter) obtained from the colony margins were transferred onto a 3- × 3-mm wound on the surface of sterilized fruit. Sterile PDA plugs were used to inoculate five control pomegranate fruits. Fruit were incubated at 22°C and 80% relative humidity in the dark. Extensive decay, similar to that observed on diseased fruits in the field, was observed on inoculated fruits 7 days after inoculation, whereas control fruits showed no decay. The pathogen was reisolated from internal rotten tissues of inoculated fruit, but not from the noninoculated control fruits. Fruit rot of pomegranate caused by B. cinerea has been reported previously in the United States (1) and China (3). However, to our knowledge, this is the first report of B. cinerea causing gray mold of pomegranate in Greece. References: (1) A. M. French. California Plant Disease Host Index. Calif. Dept. Food Agric., Sacramento, 1989. (2) W. B. Hewitt. Compendium of Grape Diseases. American Phytopathological Society, 1994. (3) Z. Zhang. Flora Fungorum Sinicorum 26:277, 2006.

First Report of Penicillium glabrum Causing Fruit Rot of Pomegranate (Punica granatum) in Greece.

During September and October of 2008 in the region of Larisa (central Greece), postharvest fruit rot was observed on pomegranate (cv. Kapmaditika), which is rapidly increasing in production in Greece, causing losses of 10 to 20% after 2 months of cold storage (5 to 6°C). Infected fruits showed green conidiophores in the calyx area, while internal symptoms consisted of soft, brown tissue that became covered with green mycelium and conidiophores. To isolate the casual agent, conidia and conidiophores were scraped aseptically from the internal fruit rot, suspended in sterile water, and streaked onto potato dextrose agar (PDA). Single hyphal tips were then transferred to new PDA plates. A fungus consistently isolated from the infected tissues was identified as Penicillium glabrum (Wehmer) Westling on the basis of morphological criteria, with conidiophores smooth or finely roughened and conidia in compact columns, glubose to subglubose, approximately 3.0 µm, with walls somewhat echinulate (1). The identification was confirmed by sequencing the internal transcribed spacer (ITS) region spanning ITS1, 5.8S, and ITS2 of the ribosomal DNA (2). The nucleotide sequence was submitted to GenBank (Accession No. FN313540). The pathogenicity of the isolated fungus was tested on five mature pomegranate fruit (cv. Kampaditika) after being surface sterilized with 5% sodium hypochlorite. A plug (5 mm in diameter) obtained from the margins of a P. glabrum colony was transferred to wounds (3 × 3 mm) made with a scalpel in the surface of fruit. Fruit inoculated with sterile PDA plugs served as controls. Fruit were incubated at 22°C and 80% relative humidity in the dark. Extensive decay, similar to that observed on diseased fruit in the field, was observed on the inoculated fruit 7 days after inoculation, whereas control fruit showed no decay. The pathogen was reisolated from inoculated fruit but not from the noninoculated fruit. To our knowledge, this is the first report of P. glabrum causing postharvest fruit rot of pomegranates in Greece. References: (1) C. Thom and K. B. Raper. Page 176 in: A Manual of the Penicillia. Williams and Wilkins, Baltimore, 1949. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Stability and fitness of anilinopyrimidine-resistant strains of Botrytis cinerea.

The fitness of anilinopyrimidine-resistant isolates of Botrytis cinerea compared with that of sensitive isolates, collected from vegetable crops in Greece during 2005, was investigated. Stability of resistance to anilinopyrimidine fungicides was determined after consecutive transfers of the fungal isolates on fungicide-free potato dextrose agar for 16 culture cycles or on fungicide-untreated cucumber seedlings for eight disease cycles. Results showed that after the consecutive transfers of the isolates either in vitro or in vivo sensitivity to cyprodinil was not changed significantly compared to the initial sensitivity in all the isolates tested, suggesting a stable genetically controlled trait. Fitness parameters measured were mycelial growth, spore production in vitro, osmotic sensitivity, virulence, spore production in vivo, percentage of spore germination, and competitive ability of the resistant isolates in four pairs with sensitive isolates both on artificial nutrient medium or on cucumber seedling plants. The measurements of the fitness components in individual isolates showed high variability within both sensitivity groups in all, except virulence, fitness components tested. As a group, resistant isolates showed significantly lower (P < 0.05) mycelial growth and virulence, while they were more osmotically sensitive than the sensitive isolates. In addition the resistant isolates showed higher (P < 0.05) spore production in vivo but there was no difference (P > 0.05) between the two sensitivity groups in spore production in vitro and in the percentage of spore germination. However, the correlation to test if there is any relationship between the values of each fitness component tested and the level of cyprodinil sensitivity of each isolate was for all, except the spore production in vivo, fitness components not significant (P > 0.05). This absence of significant correlation coefficient values suggests that the development of resistance to anilinopyrimidine fungicides did not affect the fitness of the resistant isolates. Competition of the resistant versus sensitive isolates was isolates-dependent, since in two of the isolate pairs the resistance frequency decreased significantly after five culture or disease cycles, while in the remaining two pairs resistance frequency increased significantly after five disease cycles or remained stable for one pair after five culture cycles on artificial nutrient media.

First Report of Oilseed Rape Stem Rot Caused by Sclerotinia sclerotiorum in Greece.

Oilseed rape (Brassica napus L.) was recently introduced into Greece for the production of biofuels. During May of 2007, symptoms typical of stem rot were observed on oilseed rape plants in three commercial fields in the area of Galatades-Pella, Central Macedonia, Greece. Approximately 30% of the plants were affected. Symptoms began as a chlorotic wilt on the foliage and developed into necrosis of basal stems. In the advanced stages of the disease, stems and branches became bleached and eventually died. White, as well as black, mycelium and irregularly shaped sclerotia (2 to 5 mm in diameter) were produced abundantly on and inside the affected stems. To isolate the pathogen, 20 symptomatic 6-month-old plants were collected from each field. Sclerotia were dipped in 70% ethanol, surface sterilized in 1% sodium hypochlorite for 1 min, and rinsed in sterile water. Sclerotia placed on potato dextrose agar (PDA) were incubated in the dark at 25°C for 10 days. Sclerotinia sclerotiorum (Lib.) de Bary was identified on the basis of morphological characteristics (2). To conduct pathogenicity tests, 10 6-week-old oilseed rape plants (cv. Titan) were each inoculated with a 5-mm-diameter colonized PDA disk placed in wounds made in the basal stem with a sterile scalpel. Five control plants were treated similarly except that the agar disk did not contain mycelium. Plants were then covered with a plastic bag to maintain high humidity. After 72 h, the bags were removed and the plants were maintained in a growth chamber at 23 to 25°C with a 12-h photoperiod and 75% relative humidity. Pathogenicity tests were repeated three times. Symptoms identical to those observed in the field developed within 12 days after inoculation; control plants remained healthy. The fungus was reisolated from all inoculated plants, confirming Koch's postulates. S. sclerotiorum has been reported on oilseed rape in Argentina, Australia, Brazil, Canada, the United States, and New Zealand (1). To our knowledge, this is the first report of Sclerotinia stem rot of oilseed rape in Greece. References: (1) D. F. Farr et al. Fungal Databases. Systematic Botany and Mycology Laboratory. Online publication. ARS, USDA, 2008. (2) L. M. Kohn. Phytopathology 69:881, 1979.

Baseline Sensitivity of Botrytis cinerea to Pyraclostrobin and Boscalid and Control of Anilinopyrimidine- and Benzimidazole-Resistant Strains by These Fungicides.

Fifty-five isolates of Botrytis cinerea collected from vegetable crops were used to determine the pathogen's baseline sensitivity to two new fungicides: boscalid, which inhibits the enzyme succinate dehydrogenase in the electron transport chain, and pyraclostrobin, which blocks electron transport between cytochrome b and cytochrome c1. Measurement of sensitivity to boscalid was based on both inhibition of mycelial growth and spore germination, while measurement of sensitivity to pyraclostrobin was based only on inhibition of spore germination. For both fungicides, the sensitivity distribution was a unimodal curve, with a mean EC50 value (effective concentration that reduces mycelial growth or spore germination by 50%) of 0.033 µg ml-1 for pyraclostrobin and 2.09 and 2.14 µg ml-1 for boscalid based on the inhibition of mycelial growth and spore germination, respectively. No cross-sensitivity relationship was observed between the two fungicides (r = 0.09). In addition, no cross-resistance relationship was observed between these two fungicides with other botryticides: cyprodinil, pyrimethanil, fenhexamid, fludioxonil, and iprodione. Moreover, the control efficacy of the two fungicides was tested against two anilinopyrimidine-resistant and two benzimidazole-resistant isolates, and two of wild-type sensitivity. Both pyraclostrobin and boscalid provided satisfactory control of all six isolates that was independent of the isolate sensitivity to benzimidazoles and anilinopyrimidines. In contrast, carbendazim failed to control sufficiently the benzimidazole-resistant isolates, while cyprodinil failed to provide satisfactory control of the anilinopyrimidine-resistant isolates.

First Report of Ascochyta Leaf Spot Caused by Phoma exigua var. exigua on Common Bean in Greece.

Common bean (Phaseolus vulgaris L.) is cultivated extensively in Greece for dry and fresh bean production. During 2005 and 2006, a disease with typical blight symptoms was observed occasionally on dark red kidney, brown kidney, and black bean plants in most bean-producing areas of Greece. It rarely was destructive unless the crop had been weakened by some unfavorable environmental conditions. Infected leaves had brown-to-black lesions that developed concentric zones 10 to 30 mm in diameter and also contained small, black pycnidia. Concentric dark gray-to-black lesions also appeared on branches, stems, nodes, and pods. Infected seeds turned brown to black. Plants sometimes showed defoliation and pod drop. The fungus was consistently isolated on potato dextrose agar from diseased leaves and pods and identified as Phoma exigua var. exigua Sutton and Waterstone on the basis of morphological characteristics of conidia and pycnidia (1,2). Spores were massed in pycnidia from which they were forced in long, pink tendrils under moist weather conditions. Conidia were cylindrical to oval, allantoid, hyaline, pale yellow to brown, usually one-celled, and 2 to 3 × 5 to 10 µm. To satisfy Koch's postulates, a conidial suspension (1 × 106 conidia per ml) of the fungus was sprayed onto leaves and stems of bean seedlings (first-leaf stage) (cv. Zargana Hrisoupolis). Both inoculated and control seedlings (inoculated with sterile water) were covered with plastic bags for 72 h in a greenhouse at 23°C. Inoculated plants showed characteristic symptoms of Ascochyta leaf spot 12 to 15 days after inoculation. The fungus was reisolated from lesions that developed on the leaves and stems of all inoculated plants. The pathogen is present worldwide on bean. To our knowledge, this is the first report of P. exigua var. exigua on common bean in Greece. References: (1) D. F. Farr et al. Fungal Databases. Systematic Botany and Mycology Laboratory. Online publication. ARS, USDA, 2007. (2) B. C. Sutton and J. M. Waterstone. Ascochyta phaseolorum. No. 81 in: Descriptions of Pathogenic Fungi and Bacteria. CMI/AAB, Kew, Surrey, England, 1966.

Geographical Distribution, Pathotype Characterization, and Molecular Diversity of Colletotrichum lindemuthianum in Greece and Resistance of Greek Bean Cultivars.

Thirty-five Colletotrichum lindemuthianum isolates were obtained from bean (Phaseolus vulgaris) fields in 23 prefectures of Greece during a survey conducted in 2003 and 2004. Race characterization based on the standardized set of 12 differential cultivars demonstrated the presence of races 2, 6, and 22, which are reported for first time in Greece, while race 22 is reported for the first time in the world. In addition, pathotype diversity showed significant correlation with the geographical origin of these isolates. All three races caused disease symptoms only on cultivars of Andean origin, suggesting that Greek isolates originated from South American countries. Virulence spectrum of the same isolates was also examined on a set of 30 Greek bean cultivars showing seven types of virulence. Based on their reactions to the pathogen isolates, Greek cultivars were divided into nine groups. Among these cultivars, two (Ithomi FS60 and Larisa) were resistant to all tested isolates, including the reference isolates. Molecular diversity was detected using random amplified polymorphic DNA (RAPD) primers and the restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA internal transcribed spacers, which revealed two main clusters of isolates. Thirty-two out of 35 isolates belonged to the same main cluster in both methods, indicating that Greek isolates have genotypic similarities. This study provides the information on population diversity of C. lindemuthianum, which can be useful in agricultural practices and in plant breeding programs.