Intraspecific Diversity of Monilinia fructicola and M. laxa Populations from Blossoms and Fruit of Different Hosts in Greece.

The genetic variation among 145 isolates of Monilinia fructicola and 156 isolates of M. laxa collected from two distinct regions of Greece (Imathia and Larissa) was analyzed using intersimple-sequence repeat markers. The Monilinia spp. isolates had been collected from infected fruit or blossoms of peach, apricot, sweet cherry, and plum. Calculation of Nei's gene diversity and Shannon's diversity indices showed that M. fructicola populations had higher genetic diversity compared with M. laxa populations in both regions sampled. The levels of genetic diversity were similar between populations obtained from diseased blossoms and fruit for each species and the main variances were all from within rather than between populations for the respective regions, hosts, and organ of origin. Genetic distance (Nei's analysis) was lower between peach and apricot populations than between cherry and plum populations of M. fructicola. M. fructicola isolates from peach and apricot and from sweet cherry and plum were clustered together, while M. laxa isolates clustering based on the host of origin was not possible. The analysis of index of association showed the absence of sexual recombination for both species. The derived data support the hypothesis of a long presence of M. fructicola in Greece, and provide evidence of specialization of M. fructicola populations based on their host of origin.

Fitness and competitive ability of Botrytis cinerea field isolates with dual resistance to SDHI and QoI fungicides, associated with several sdhB and the cytb G143A mutations.

Respiration inhibitors such as the succinate dehydrogenase inhibitors (SDHIs) and the quinone outside inhibitors (QoIs) are fungicide classes with increasing relevance in gray mold control. However, recent studies have shown that dual resistance to both fungicide classes is a common trait in Botrytis cinerea populations from several hosts throughout the world. Resistance of B. cinerea to SDHIs is associated with several mutations in the sdhB, sdhC, and sdhD genes, while resistance to QoIs, in most cases, is associated with the G143A mutation in the cytb gene. The objective of the current study was to investigate the fitness and the competitive ability of B. cinerea field strains possessing one of the H272Y/R/L, N230I, or P225F sdhB substitutions and the G143A mutation of cytb. Fitness parameters measured were (i) mycelial growth and conidia germination in vitro, (ii) aggressiveness and sporulation capacity in vivo, (iii) sclerotia production in vitro and sclerotia viability under different storage conditions, and (iv) sensitivity to oxidative stress imposed by diquat treatments. The competitive ability of the resistant isolates was measured in the absence and presence of the SDHI fungicides boscalid and fluopyram selection pressure. The measurements of individual fitness components showed that the H272R/G143A isolates had the lower differences compared with the sensitive isolates. In contrast, the groups of H272Y/L/G143A, N230I/G143A, and P225F/G143A isolates showed reduced fitness values compared with the sensitive isolates. Isolates possessing only the cytb G143A substitution did not show any fitness cost. The competition experiments showed that, in the absence of fungicide selection pressure, after four disease cycles on apple fruit, the sensitive isolates dominated in the population in all the mixtures tested. In contrast, when the competition experiment was conducted under the selection pressure of boscalid, a gradual decrease in the frequency of sensitive isolates was observed, whereas the frequency of H272L and P225F isolates was increased. When the competition experiment was conducted in the presence of fluopyram, the sensitive isolates were eliminated even after the first disease cycle and the P225F mutants dominated in the population. Such results suggest that the sdhB mutations may have adverse effects on the mutants. The observed dominance of sensitive isolates in the competition experiments conducted in the absence of fungicides suggest that the application of SDHIs in alternation schemes may delay the selection or reduce the frequency of SDHI-resistant mutants.

Vineyard practices reduce the incidence of Aspergillus spp. and alter the composition of carposphere microbiome in grapes (Vitis vinifera L.).

Going through the new transitioning era of the "European Green Deal," the search for alternative, non-chemical, disease control methods is essential. Aspergillus bunch rot is considered one of the most important diseases of grapevines resulting in severe yield losses and, major qualitative deterioration of grape products due to the production of mycotoxins. We investigated, in a two-year field study, the impact of agronomic practices like defoliation to enhance grape microclimate (DF), pruning method to reduce grape bunch density (LBD), and irrigation cut-off (NIR), at three developmental stages of grapevine (Pea size berry, Veraison, and Harvest), on (i) grape composition (titratable acidity, pH, and total soluble solids), (ii) on the frequency of occurrence of Aspergillus on grape berries, and (iii) on the overall composition of grape carposphere microbiome. The density of Aspergillus on grape berries was significantly reduced by the applied management practices (DF, LBD, and NIR). Amplicon sequencing analysis showed that both the phenological stage and the agronomic practices employed (particularly NIR and DF) imposed significant changes in the α-diversity and ß-diversity of the grape carposphere bacterial and fungal communities. The NIR, LBD, and DF treatments which supported lower Aspergillus populations, network analysis revealed negative co-occurrence patterns between Aspergillus and several bacterial genera (Streptococcus, Rhodococcus, and Melitangium) reported to have antifungal properties suggesting potential natural attenuation mechanisms for the control of Aspergillus. Overall, our study (i) showed that the application of halting of irrigation and thinning of leaves and grape bunches, reduce the occurrence of Aspergillus and hence the incidence of Aspergillus Bunch rot disease and (ii) identified preliminary evidence for interactions of Aspergillus with members of the epiphytic grape bacterial communities that might be involved in the suppression of Aspergilli, an observation which will be further pursued in following studies in the quest for the discovery of novel biological control agents.

Competitive Ability and Fitness of Alternaria alternata Isolates Resistant to QoI Fungicides.

Fungicides that act as quinone outside inhibitors (QoIs) constitute a fungicide group extensively used against Alternaria late blight of pistachio caused by Alternaria spp. However, developement of resistance to this fungicide class constitutes an important threat for the succesful control of the disease. This study was conducted to determine whether development of resistance to QoIs is associated with a fitness cost, by measuring several biological and epidemiological parameters and estimating the competitive ability in four QoI-resistant and four QoI-sensitive Alternaria alternata isolates. Fitness parameters measured were mycelial growth and spore production in vitro, disease latent period, aggressiveness, and spore production on detached pistachio leaves. The competitive ability of resistant isolates was assessed in coinoculation experiments with sensitive isolates on detached pistachio leaves, using a real-time polymerase chain reaction assay technique. Fitness parameters between grouped QoI-resistant and QoI-sensitive isolates were not significantly different (P = 0.13, 0.21, 0.31, and 0.27 for sporulation in vitro, mycelial growth, incubation period, and sporulation in vivo, respectively), while resistant isolates, as a group, showed a higher aggressiveness (P = 0.01) compared with the sensitive isolates. The data indicate that the resistant strains did not account for a fitness cost compared with the sensitive ones under the conditions of testing. The outcome of the competition experiments was isolate dependent. In two pairs, the resistance frequencies increased whereas, in the remaining two pairs of isolates, resistance frequency decreased, suggesting that the resistant isolates were competitive similarly to the sensitive isolates.

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.

Characterization of Rhizoctonia spp. Isolates Associated with Damping-Off Disease in Cotton and Tobacco Seedlings in Greece.

Isolates of Rhizoctonia spp. were obtained during the spring of 2007 from diseased cotton and tobacco seedlings showing damping-off symptoms. The sampled fields were located in the main cotton- and tobacco-cultivating regions of central and northern Greece. Among the 79 isolates obtained from cotton plants, 17 were binucleate and 62 were multinucleate whereas, among the 89 isolates obtained from tobacco plants, 87 were multinucleate and only 2 were binucleate. Characterization of anastomosis groups (AGs) was performed with hyphal anastomosis reactions using tester isolates of known AG groups. Anastomosis reactions in cotton mutlinucleate isolates showed that 54 of them belonged in Rhizoctonia solani AG-4, 6 in AG-7, 1 in AG-2, and 1 in AG-3. In the 87 tobacco multinucleate isolates, anastomosis reactions showed that 70 of them belonged in R. solani AG-2, 16 in AG-4, and 1 in AG-5. In addition, molecular characterization was carried out using the specific ribosomal DNA internal transcribed spacer region, in a randomly selected number of isolates. In cotton, the most prevalent AG was AG-4, with 18 isolates to the subgroup HG-I, 1 isolate to the subgroup HG-II, and 7 isolates to the subgroup HG-III, followed by AG-7 (7 isolates), AG-2-1 (1 isolate), and AG-3 (1 isolate). In tobacco, the most prevalent group was AG-2-1 (70 isolates), followed by AG-4 (6 isolates to the subgroup HG-I and 5 isolates to the subgroup HG-III) and a single isolate belonging to AG-5. Phylogenetic analysis showed that the isolates were distinctly separated based on their AG types. Pathogenicity and aggressiveness of the isolates to several hosts was determined. AG-4 isolates from either cotton or tobacco were the most aggressive on the hosts tested, while AG-2-1 isolates were of moderate aggressiveness and were not pathogenic on barley.

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.

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 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.

Resistance of Botrytis cinerea Isolates from Vegetable Crops to Anilinopyrimidine, Phenylpyrrole, Hydroxyanilide, Benzimidazole, and Dicarboximide Fungicides.

During February 2005, 55 single-spore isolates of Botrytis cinerea were collected at the end of the season from vegetable crops grown in 18 greenhouses on the island of Crete, Greece. They were tested for sensitivity to the anilinopyrimidine fungicides pyrimethanil and cyprodinil, the hydroxyanilide fungicide fenhexamid, the phenylpyrrole fungicide fludioxonil, the dicarboximide fungicide iprodione, and the benzimidazole fungicide carbendazim. Results of the study showed the existence of benzimidazole- and dicarboximide-resistant strains at frequencies of 61.8 and 18%, respectively. Moreover, for first time, the development of resistance to anilinopyrimidine fungicides by B. cinerea was detected in greenhouse vegetable crops on the island of Crete. High resistance frequencies of 49.1 and 57.4% were observed for pyrimethanil and cyprodinil, respectively. In addition, one isolate was found to be resistant to the hydroxyanilide fungicide fenhexamid, while no strains resistant to the phenylpyrrole fungicide were detected. Among the 55 isolates tested, 13 were resistant only to carbendazim, 6 were resistant only to anilinopyrimidines, 3 were resistant to both benzimidazoles and dicarboximides, 17 were resistant to both benzimidazoles and anilinopyrimidines, 6 were resistant to both dicarboximides and anilinopyrimidines, 1 was simultaneously resistant to benzimidazoles, dicarboximides, and anilinopyrimidines, 1 was resistant to both anilinopyrimidines and hydroxyanilides, and 8 were sensitive to all fungicides tested. A strong cross-resistance relationship was found between the two anilinopyrimidine fungicides tested when log transformed EC50 values of the isolates were subjected to a linear regression analysis (r = 0.71). Despite the detection of several phenotypes with simultaneous resistance to chemically unrelated active ingredients, in none of the remaining possible fungicide pairs was there observed any kind of cross-resistance relationship.

Comparative Efficacy, Selection of Effective Partners, and Application Time of Strobilurin Fungicides for Control of Cercospora Leaf Spot of Sugar Beet.

In this study, we attempt to optimize the use of strobilurin fungicides by testing the efficacy of azoxystrobin, kresoxim-methyl, pyraclostrobin, and trifloxystrobin under field conditions, by testing for the most efficient partners in fungicide mixtures, and by testing control efficacy of strobilurin fungicides applied at several application times to determine the better options for disease management. Results showed that trifloxystrobin was the most efficient strobilurin fungicide, followed by pyraclostrobin. Azoxystrobin provided a modest to poor control efficacy, whereas kresoxim-methyl provided only poor disease control efficacy. Mixtures of azoxystrobin and trifloxystrobin with either chlorothalonil or maneb and difenoconazole or flutriafol were tested for their efficacy in controlling the disease. The results showed that the azoxystrobin-containing mixtures provided significantly better control compared with that obtained by single applications of each mixture component. The mixtures of trifloxystrobin with maneb or with difenoconazole or flutriafol provided control efficacy similar to that obtained by single applications of trifloxystrobin, whereas the mixture of trifloxystrobin and chlorothalonil provided significantly lower control efficacy compared with the other trifloxystrobin-containing mixtures tested. For both strobilurin fungicides tested, the calculated ratio between the observed and the expected control efficacy ranged around the value of 1, suggesting additive interactions between the mixtures' components. To determine the most appropriate time for strobilurin fungicides application, trifloxystrobin was applied as the first two, the middle two, or the final two consecutive treatments of six fungicide applications. The remaining fungicide treatments in the spray schedules were carried out by applying the systemic fungicide difenoconazole. Results showed that a higher control efficacy was obtained when trifloxystrobin was applied in either of the earlier applications.

First Report of Phomopsis Fruit Decay on Apple Caused by Phomopsis mali in Greece.

From 2002 to 2005, a previously unreported disease causing significant yield losses was observed on apple fruits (cv. Red Chief) in the region of Imathia in northern Greece. Almost all apple orchards in that area, cultivated with Red Chief, showed disease symptoms on 3 to 10% of the fruits. Diseased fruits showed irregularly shaped, water-soaked areas on the skin and extensive decay internally. In most of the fruits, decay appeared to initiate internally from the calyx tube. Infected fruits remained firm during the early stages of decay. Fungal isolates obtained from small pieces of decayed tissue on potato dextrose agar (PDA) medium were identified as Phomopsis mali Roberts on the basis of morphological characteristics (2). Cultures grew rapidly on PDA at 22°C in the dark. They were initially white, but approximately 30 days after inoculation they turned gray because of the formation of pycnidia that contained α- and ß-spores. α -Spores were short and elliptical in shape (8 to 10 × 2 to 3 µm) while ß-spores were long (22 to 25 × 1 to 2 µm). Pathogenicity of the isolated cultures was tested by wound inoculating five mature apple fruits (cv. Red Chief) after surface sterilization with 0.5% NaOCl. PDA plugs, 5 mm in diameter with actively growing mycelium, were transferred into the flesh of the fruits. Sterile PDA plugs were used to inoculate five control apple fruits. Inoculated fruits were kept at 23°C for 10 days in the growth chamber. Extensive decay, similar to that observed on diseased fruits in the field, was observed on the inoculated fruits, whereas control fruits showed no decay. P. mali was reisolated from the decayed tissues. Commercial losses due to fruit decay caused by the pathogen have previously been reported in the United States and Northern Ireland (1). To our knowledge, this is the first report of Phomopsis fruit decay on apples in Greece. References: (1) A. C. Jones and H. S. Aldwinckle eds. Compendium of Apple and Pear Diseases. The American Phytopathological Society. St. Paul, MN, 1990. (2) D. A. Rosenberger and T. J. Burr.

Control of Benzimidazole- and DMI-Resistant Strains of Cercospora beticola with Strobilurin Fungicides.

The control efficacy of two new strobilurin fungicides, trifloxystrobin and pyraclostrobin, against Cercospora beticola isolates resistant and sensitive to sterol demethylation-inhibiting (DMI) fungicides and benzimidazole fungicides and the effects on evolution of resistance were tested in the current study. Control efficacy of strobilurin fungicides was measured using three C. beticola isolates, one DMI-resistant (DMIR), one benzimidazole-resistant (BENR), and one of wild-type sensitivity (WCB). Both pyraclostrobin and trifloxystrobin provided satisfactory control of all the three isolates used in the study, when applied at 5 µg ml-1 and very high levels of control when applied at 10 µg ml-1. Control was independent of the isolate sensitivity to benomyl and difenoconazole. In contrast, benomyl applied at 10 µg ml-1 failed to control sufficiently the benzimidazole-resistant isolate, whereas difenoconazole applied at either 5 or 10 µg ml-1 failed to provide satisfactory control of the DMI-resistant isolate of the pathogen. The effects of strobilurin fungicide applications on the evolution of resistance to benzimidazole and DMI fungicides were tested under field conditions in a 2-year experiment (2003 to 2004). Applications of either trifloxystrobin or pyraclostrobin provided high levels of disease control during both years of the study, whereas applications of either benomyl or difenoconazole provided a moderate control efficacy. Measurements of resistance frequencies to benomyl and to difenoconazole showed that successive applications of benomyl tended to select for high frequencies of benzimidazole-resistant phenotypes, whereas successive applications of difenoconazole tended to select for high frequencies of DMI-resistant phenotypes. In contrast, applications of either trifloxystrobin or pyraclostrobin prevented an increase of benzimidazole- or DMI-resistant phenotypes compared with the plots treated with benomyl or difenoconazole, respectively, and decreased frequency of resistance compared with untreated control plots.

First Report of Phytophthora Root Rot of Sugar Beet, Caused by Phytophthora cryptogea, in Greece.

A severe rot of sugar beet roots was observed in the Amyndeon area of Greece during summer 1998. Infected plants initially showed a temporary wilt, which became permanent, and finally died. Slightly diseased roots showed necrotic spots toward the base, whereas more heavily diseased roots showed a more extensive wet rot that extended upward. Feeder roots also were infected and reduced in number because of decay. Rotted tissue was brown with a distinguishing black margin. In most of the isolations, carried out on potato dextrose agar (PDA), the pathogen obtained was identified as Phytophthora cryptogea Pethybr. & Lafferty Mycelium consisted of fairly uniform, fine hyphae that showed a slightly floral growth pattern. In autoclaved soil-extract medium, chains or clusters of hyphal swellings (average 12 µm diameter) formed. Sporangia were not produced on solid media but were abundant in soil-extract medium. Sporangia were oval to obpyriform in shape, nonpapillate with rounded bases, and varied in size (39 to 80 × 24 to 40 µm). Oospores were plerotic, thick-walled, and averaged 25 µm in diameter. The isolated pathogen, cultured on PDA, could not grow at all at 36°C. The closely related species P. drechsleri Tucker has been reported to cause similar root rot symptoms on sugar beet (3). However, P. drechsleri grows well at 36°C, while P. cryptogea cannot grow at this temperature; this is the major distinguishing feature that separates the two species (1). To test the pathogenicity of the organism, surface-sterilized sugar beet roots (cv. Rizor) were inoculated with 5-mm-diameter PDA plugs containing actively growing mycelium. Sterile PDA plugs were used to inoculate control sugar beet roots. Inoculated roots were kept at 27°C in the dark for 10 days. Extensive decay of inoculated roots developed, similar to decay observed in the field, whereas control roots showed no decay. P. cryptogea was reisolated from rotted tissues. This pathogen has been recognized previously as a cause of root rot of sugar beet in Japan (1) and Wyoming (2). This is the first report of Phytophthora root rot of sugar beet in Greece. References: (1) D. C. Erwin and O. K. Ribeiro. 1996. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN. (2) P. C. Vincelli et. al. Plant Dis. 74:614, 1990. (3) E. D. Whitnew and J. E. Duffus, eds. 1986. Compendium of Beet Diseases and Insects. The American Phytopathological Society, St. Paul, MN.

First Report of Verticillium Wilt of Sugar Beet, Caused by Verticillium dahliae, in Greece.

Wilting sugar beet plants (Beta vulgaris L.) were observed in fields in the Larissa area of central Greece during the summers of 1997 and 1998. Diseased plants, showing general yellowing and epinasty, were sporadically distributed in the fields. As symptoms progressed, the outer leaves wilted and became desiccated. Inner leaves showed marginal and interveinal yellowing. These areas later turned brown and became necrotic. Longitudinal sections of the roots of diseased plants displayed browning of vascular tissues. Fungal isolates obtained from discolored vascular tissues on potato dextrose agar (PDA) medium were identified as Verticillium dahliae based on morphological features (1). Cultures grew moderately fast on PDA at 23°C. Mycelia were hyaline and white to cream colored, becoming black with formation of microsclerotia. Conidiophores were hyaline and verticillately branched, with three to four phialides at each node. Conidia borne on phialides were ellipsoidal to short and cylindrical and mainly one-celled (2.5 to 8 × 1.4 to 3.2 µm). Microsclerotia began to form in 6- to 7-day-old cultures and were dark brown to black and varied in shape and size (25 × 50 to 100 µm diameter). Pathogenicity tests were carried out using the root-dip technique. Two-week-old seedlings (cv. Rizor) were inoculated by dipping roots in an aqueous suspension of 108 conidia per ml for 1 min. Inocula were obtained from 2-week-old cultures grown on PDA at 21°C by adding sterile water to the petri dish, gently shaking to detach conidia from the conidiophores, and filtering through a doublelayer of sterile cheesecloth. Roots of control plants were dipped in distilled water, and seedlings were transplanted to pots and placed in a growth chamber at 23°C with a 12-h photoperiod. Inoculated plants exhibited wilted leaves with interveinal yellowing ≈30 days after inoculation; symptoms were not observed on control plants. V. dahliae was reisolated from artificially inoculated plants. Measurements of yield parameters in healthy and diseased plants showed that the sugar content of diseased roots was significantly reduced, whereas root weight was not affected. Such results agree with a previous report on the effects of the disease on yield parameters (2). However, the disease is of minor importance in Greece mainly because of the low number of infected plants in the fields. This is the first report of Verticillium wilt of sugar beet in Greece. References: (1) Anonymous. 1971. Verticillium dahliae. No 256: Descriptions of Plant Pathogenic Fungi and Bacteria. Common w. Mycol. Inst., Kew, England. (2) J. O. Gaskill and W. A. Krentzer. Phytopathology 30:769, 1940.

First Report of Charcoal Rot of Sugar Beet Caused by Macrophomina phaseolina in Greece.

During the summer of 2000 in the Amyndeon area of northern Greece, sugar beet (Beta vulgaris L.) roots with rot symptoms were observed in many fields. Initially, the plants wilted, and leaves soon turned brown and died. Diseased plants appeared in patches in the field. Brown-black lesions were observed in the external part of the root crown while yellow-mustard colored lesions occurred internally. In advanced stages of decay, masses of sclerotia formed in rotted cavities and roots became mummified. Macrophomina phaseolina (Tassi) Goid. (1) was isolated on potato dextrose agar (PDA) from 30 rotted roots collected in five fields. Cultures produced dark multi-septate mycelium and sclerotia, which were black, smooth, spherical to irregular in shape, and varied in size from 100 µm to 1mm in diameter. Five isolates were evaluated for pathogenicity on surface-sterilized 16-week-old sugar beet roots (cv. Rizor) by placing a 5-mm-diameter PDA plug of actively growing mycelium in wounds made with a sterile knife. Sterile PDA plugs were placed in wounds made in control beet roots. Ten roots were inoculated per isolate. Roots were kept at 25°C in the dark for 10 days. Extensive decay of inoculated roots developed, similar to decay observed in the field, and M. phaseolina was reisolated from rotted tissue. Control roots showed no decay. This pathogen has been previously reported as a root rot pathogen of sugar beet in California, India, and countries of the former USSR. Charcoal rot is of minor economic importance since M. phaseolina attacks mainly weakened plants under conditions of high temperature (2). To our knowledge, this is the first report of charcoal root rot of sugar beet in Greece. References: (1) Anonymous. Macrophomina phaseolina. No. 275 in: Descriptions of Plant Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1970. (2) J. E. Duffus and E. G. Ruppel. Diseases. Page 347 in: The Sugar Beet Crop. Science into Practice. D. A. Cooke and R. K. Scott eds. Chapman and Hall, NY, 1993.