A Novel Seimatosporium and Other Sporocadaceae Species Associated with Grapevine Trunk Diseases in Cyprus

Besides well-known grapevine trunk disease (GTD)-related pathogens, there is an increased interest in wood-colonizing fungi that infect grapevines. During 2017-2018, a survey was conducted in Cyprus and wood samples were collected from vines exhibiting typical GTD symptoms. Based on morphological and multilocus phylogenetic analyses (ITS, LSU, bt2, tef1-a), four species in the Sporocadaceae family were described and typified; two in the genus of Seimatosporium: Seim. cyprium sp. nov. and Seim. vitis-viniferae and two in Sporocadus: Spo. kurdistanicus and Spo. rosigena. The teleomorph of Seim. cyprium sp. nov. was also described. Pathogenicity trials with representative isolates of each species were performed on woody stems of two-year-old potted grapevines for 12 months under field conditions. All isolates were pathogenic, causing dark brown to black vascular discoloration, extending upward and downward from the inoculation point. Sporocadus isolates were significantly more aggressive than Seimatosporium with lesion lengths ranging from 9.24 to 6.90 and 4.13 to 4.00 cm, respectively. Successful re-isolations were also evident for all species and isolates. Seim. cyprium sp. nov. is a newly described species, while Spo. kurdistanicus and Spo. rosigena are reported for the first time in Europe on Vitis vinifera, suggesting the potential role of Sporocadaceae in the GTDs complex

"Jumping Jack": Genomic Microsatellites Underscore the Distinctiveness of Closely Related Pseudoperonospora cubensis and Pseudoperonospora humuli and Provide New Insights Into Their Evolutionary Past

Downy mildews caused by obligate biotrophic oomycetes result in severe crop losses worldwide. Among these pathogens, Pseudoperonospora cubensis and P. humuli, two closely related oomycetes, adversely affect cucurbits and hop, respectively. Discordant hypotheses concerning their taxonomic relationships have been proposed based on host-pathogen interactions and specificity evidence and gene sequences of a few individuals, but population genetics evidence supporting these scenarios is missing. Furthermore, nuclear and mitochondrial regions of both pathogens have been analyzed using microsatellites and phylogenetically informative molecular markers, but extensive comparative population genetics research has not been done. Here, we genotyped 138 current and historical herbarium specimens of those two taxa using microsatellites (SSRs). Our goals were to assess genetic diversity and spatial distribution, to infer the evolutionary history of P. cubensis and P. humuli, and to visualize genome-scale organizational relationship between both pathogens. High genetic diversity, modest gene flow, and presence of population structure, particularly in P. cubensis, were observed. When tested for cross-amplification, 20 out of 27 P. cubensis-derived gSSRs cross-amplified DNA of P. humuli individuals, but few amplified DNA of downy mildew pathogens from related genera. Collectively, our analyses provided a definite argument for the hypothesis that both pathogens are distinct species, and suggested further speciation in the P. cubensis complex

Global Geographic Distribution and Host Range of Fusarium circinatum, the Causal Agent of Pine Pitch Canker

Funding: This study was financially supported by COST Action FP1406 (PINESTRENGTH), the Estonian Science Foundation grant PSG136, the Forestry Commission, United Kingdom, the Phytophthora Research Centre Reg. No. CZ.02.1.01/0.0/0.0/15_003/0000453, a project co-financed by the European Regional Development Fund. ANSES is supported by a grant managed by the French National Research Agency (ANR) as part of the “Investissements d’Avenir” programme (ANR-11-LABX-0002-01, Laboratory of ExcellenceARBRE). SW was partly supported by BBSRC Grant reference BB/L012251/1 “Promoting resilience of UK tree species to novel pests & pathogens: ecological & evolutionary solutions (PROTREE)” jointly funded by BBSRC, Defra, ESRC, the Forestry Commission, NERC and the Scottish Government, under the Tree Health and Plant Biosecurity Initiative. Annual surveys in Switzerland were financially supported by the Swiss Federal Office for the Environment FOEN. Acknowledgments: Andrea Kunova and Cristina Pizzatti are acknowledged for the assistance in the sampling. Thanks are due to Dina Ribeiro and Helena Marques from ICNF-Portuguese Forest Authority for providing location coordinates. We thank three anonymous reviwers for valuable corrections and suggestions.Peer reviewedPublisher PD

First report of cytospora punicae causing trunk canker of pomegranate (punica granatum) in Cyprus

Organized cultivation of pomegranate (Punica granatum L.) in Cyprus is relatively recent and accelerating, with approximately 420 acres currently under cultivation. In August 2013, a pomegranate orchard cv. Ermioni with severe trunk malformations, located in the mountainous region of Pitsilia (elevation 1100 m) in the province of Limassol, Cyprus, was brought to our attention. Trees were 2 years old and almost 30% of trees exhibited on their trunk longitudinal sunken cankers, ranging from 5 to 30 cm long. At the canker site, the bark was torn open, rendering exposed sapwood reddish-brown. The foliage was yellowish, while in a few cases the trunk was girdled, causing tree death, while no canker formation was found on the tree branches. Fungal isolations were performed by plating plant tissue from discolored sapwood on potato dextrose agar (PDA) amended with streptomycin sulfate at 100 μg/ml, and representative isolates were subcultured onto PDA at 25°C. Developed thalli were comprised of septate hyphae that were whitish and progressively turned olive green to dark brown with maturity. Dark-brown pycnidia (n = 25) were obvious after 2 weeks and measured 248.6 to 580.4 μm in diameter (mean 389.4 μm; SD ± 90.9 μm). Conidia (n = 50) were hyaline, aseptate, and allantoid, ranging from 4.5 to 5.4 μm long (mean = 4.8 μM; SD ± 0.4 μm) × 0.9 to 1.9 μm wide (mean = 1.4 μm; SD ± 0.3 μm). The internal transcribed spacer region (ITS1-5.8S-ITS2) of rDNA and the translation elongation factor 1-alpha gene (EF1-α) were amplified and sequenced. Consensus sequences were deposited in GenBank (Accession Nos. KR020716 and KR020717) and BLAST alignment in the NCBI database revealed high homology (99 to 100%) to other previously reported for Cytospora punicae Sacc. (Saccardo 1884) (Accession Nos. KJ621688, KJ621689, KJ621684, and JX438568). In October 2013, 10 one-year-old branches of P. granatum cv. Ermioni were inoculated by placing mycelium plugs from actively growing colonies on PDA into wounds made by removing the bark with a cork borer, while 5 control branches were inoculated with sterile PDA plugs. Inoculations sites were covered with Vaseline and wrapped with parafilm to prevent desiccation. Six months later, branches were examined for canker development. C. punicae-inoculated branches developed longer cankers (25.8 cm) compared with the control branches (0.6 cm). The fungus was reisolated only from inoculated branches, whereas isolations from nonexpanding wounds were negative, indicating that the fungal mycelium placed in the wounds was responsible for canker formation. Koch’s postulates were fulfilled. C. punicae has been previously described as a stem, twig, and branch pathogen causing canker and dieback on pomegranate trees (Palou et al. 2013; Peduto Hand et al. 2014). The opportunistic nature of Cytospora spp. fungi is well described and canker development has been reported to begin at trees predisposed to inciting factors causing acute stress, including drought, flood, insect damage, and frost (Guyon et al. 1996). Thus, further research to determine the predisposing factors for trunk canker development in pomegranates by C. punicae may be considered in the near future. To our knowledge, this is the first report of the aforementioned pathosystem in Cyprus

First report of powdery mildew of Sideritis perfoliata caused by Neoerysiphe galeopsidis in Cyprus

The genus Sideritis L. (Lamiaceae) comprises approximately 150 perennial and annual species, mainly in the Mediterranean Basin, and has been used traditionally for culinary and therapeutical purposes (González-Burgos et al. 2011). Medicinal and aromatic plants (MAPs) constitute a dynamic sector for Cyprus due to the ideal prevailing climatic conditions and the relatively limited required crop resources. Currently, MAPs are grown on approximately 160 ha in Cyprus exported mainly to European markets. During the winter of 2014, 0.4 ha of Sideritis perfoliata plants grown on an organic farm near Nicosia were severely affected by a foliar disease that rendered the crop unmarketable. Symptoms included yellow, spotty discoloration of infected leaves that resulted in wilting and necrosis. Signs resembled those of a powdery mildew. Mycelium was sparse and not easily noticed on the upper leaf area due to the density of trichomes. Conidiophores were hyaline and straight, with 2 to 5 hyaline, doliform conidia that lacked fibrosin bodies and ranged from 35 to 49 μm (mean ± standard deviation of 42 ± 4 μm) × 17 to 25 μm wide (mean = 21 ± 3 μm). Chasmothecia were abundant, partly clustered, yellowish to dark brown, globose, 102 to 203 μm in diameter (157 ± 38 μm) each containing 5 to 8 asci. Numerous hyaline, septate, mycelioid appendages, generally simple and rarely branched, were observed on the chasmothecia. Asci were stalked, kidney-shaped, and ranged from 42 to 90 μm (58 ± 17 μm) × 18 to 35 μm (25 ± 5 μm). No mature ascospores were found on the examined specimens. The microscopic features were consistent with those of Neoerysiphe galeopsidis (DC.) U. Braun (Braun and Cook 2012). For molecular identification, fungal genomic DNA of isolate NG_CY1 was extracted from infected tissue and part of the internal transcribed spacer (ITS) ribosomal DNA (rDNA) region was amplified using primers ITS5 and P3 according to Takamatsu et al. (2009). The PCR product was sequenced. BLAST analysis of the 548-bp fragment (GenBank Accession No. KR006933) showed ≥99% similarity to N. galeopsidis ITS sequences in the NCBI database (AB498942, AB498949, DQ359698, and AB022370). Pathogenicity was confirmed by gently pressing diseased leaves onto leaves of five healthy plants of S. perfoliata. An equal number of noninoculated plants served as control plants. All plants were covered with polyethylene bags for 2 days and incubated in a growth chamber at 24°C, after which they were uncovered and maintained at the same temperature with a 12-h photoperiod. Powdery mildew signs and symptoms were first observed approximately a week later. The fungus that developed was morphologically identical to that observed on the original diseased plants, while no symptoms developed on the control plants, therefore, Koch’s postulates were fulfilled. Although N. galeopsidis has been reported on plants from 12 families, mainly species in the Lamiaceae, and on other Sideritis species (Farr and Rossman 2015, Pastircakova et al. 2008), to our knowledge this is the first report of powdery mildew caused by N. galeopsidis on S. perfoliata in Cyprus

Baseline sensitivities for new postharvest fungicides against Penicillium spp. on citrus and multiple resistance evaluations in P. digitatum

For the first time in over 25 years, three new fungicides (azoxystrobin, fludioxonil, and pyrimethanil), all belonging to different chemical classes, are being registered for postharvest use against Penicillium decays of citrus fruit in the United States. Baseline sensitivities of Penicillium digitatum and P. italicum were developed using isolates collected before the commercial use of these new fungicides. In a comparison of methods, EC50 values obtained using the spiral gradient dilution method were very similar to those obtained using traditional agar dilutions of fungicides. For azoxystrobin, the addition of salicylhydroxamic acid (SHAM) did not significantly affect EC 50 values for mycelial growth of both species. In additional studies on conidial germination of P. digitatum, SHAM significantly reduced EC 50 values for azoxystrobin. For pyrimethanil, the mean EC 50 value for mycelial growth obtained using a minimum growth medium for anilinopyrimidine fungicides was significantly lower but comparable to values obtained when using potato dextrose agar. For mycelial growth of P. digitatum, mean EC50 values were 0.014, 0.025, and 0.313 μg/ml, whereas for conidial germination, they were 0.074, 0.163, and 1.195 μg/ml for azoxystrobin, fludioxonil, and pyrimethanil, respectively. For P. italicum, mean EC50 values for mycelial growth for fludioxonil and pyrimethanil were 0.005 and 0.040 μg/ml, respectively. For azoxystrobin, the mean EC 50 value for mycelial growth for 33 isolates was 0.029 μg/ml. Four isolates had EC50 values ≥0.772 μg/ml and were considered part of a resistant subpopulation. Multiple resistance between the older and new postharvest fungicide classes on citrus was not detected in P. digitatum, and all isolates that were sensitive or resistant to imazalil or thiabendazole were sensitive to the new compounds. This information is important for monitoring populations of P. digitatum, where resistance against the older fungicides has commonly develope

Characterization of Rhizoctonia solani Associated with Black Scurf in Cyprus

During 2011, 96 sclerotial isolates of Rhizoctonia solani were collected from potato tubers from all main potato-cultivating regions of Cyprus. All isolates were found to be multinucleate. Characterization of anastomosis groups (AG) based on hyphal anastomosis reactions showed that 91 isolates belonged to AG3 and 5 to AG4. Sequence analysis of the internal transcribed spacer (ITS) regions (ITS1 and ITS2) of ribosomal DNA (rDNA) of 68 isolates confirmed the prevalence of AG3. In addition, phylogenetic analysis found that AG3 isolates were of the potato type, distinctly separated from the AG3 tobacco type, while AG4 isolates were separated into two different subgroups (HGI and HGII). Temperature studies showed that isolates belonging to both AG4 subgroups had significantly higher optimum growth temperatures compared with AG3. In vitro sensitivities to the fungicide pencycuron, in terms of concentrations where 50% growth inhibition was observed, ranged from 0.012 to 0.222 mu g/ml. Pathogenicity and aggressiveness of the isolates was determined on 'Annabelle' potato sprouts and seedlings of a number of selected hosts, based on crop rotations followed in Cyprus. The majority of the isolates were pathogenic to potato sprouts, with disease severity (DS) values ranging from 0 to 88%. Mean DS values were statistically different among AG and subgroups, with AG4-HGI (69.25%) and AG4-HGII (3.12%) being the most and least aggressive, respectively. However, AG4-HGII isolates were the most aggressive in all rotational hosts tested, while AG3 isolates were the least aggressive. More specifically, the highest DS levels by AG4-HGI were recorded to barley, by AG4-HGII to lettuce and melon, and by AG3 isolates to vetch. This is the first comprehensive study to elucidate the AG composition, pathogenicity and other biological aspects of R. solani isolates associated with potato black scurf in Cyprus

Comparative efficacy of the new postharvest fungicides azoxystrobin, fludioxonil, and pyrimethanil for managing citrus green mold

Three new fungicides (i.e., azoxystrobin, fludioxonil, and pyrimethanil) are currently being introduced for postharvest management of citrus green mold in the United States. The effectiveness of each fungicide was evaluated when applied alone (at 1,000 to 1,200 mg/liter) or in mixtures (at 500 mg/liter each component) to lemon fruit that were wound-inoculated with imazalil/thiabendazole (TBZ)-sensitive or -resistant isolates of Penicillium digitatum. In laboratory studies when aqueous fungicide solutions were applied 9 to 21 h after inoculation, pyrimethanil showed the highest level of green mold control. The efficacy of fludioxonil and azoxystrobin was very high at the early timings, but decreased as time after inoculation increased. Differences in fungicide performance were not due to multiple fungicide resistance, but more likely due to differences in fungicide mobility in fruit tissue. Azoxystrobin-fludioxonil mixtures were significantly more effective when compared to single-fungicide treatments. Mixtures of imazalil with pyrimethanil were the most effective in controlling decay. The efficacy of all fungicides was significantly lower when mixed into a packing fruit coating as compared to aqueous or storage fruit coating applications. In laboratory and packingline studies, the lowest incidence of green mold decay was obtained when azoxystrobin-fludioxonil and imazalil-pyrimethanil were applied as aqueous solutions that were followed by a fruit coating. Among the new fungicides, azoxystrobin and fludioxonil applied in water or storage fruit coating, respectively, provided the best anti-sporulation activity. Storage fruit coating improved the activity of both fungicides. Pyrimethanil was the least effective fungicide in suppressing sporulation of the pathogen on decaying fruit. Overall, among the mixtures, azoxystrobin-fludioxonil and TBZ-fludioxonil had high anti-sporulation activity in aqueous and storage fruit coating applications. New integrated management programs should be based on monitoring of fungicide sensitivities in pathogen populations, rotating mixtures of products with different modes of action, and using appropriate fungicide application strategie

Characterization of Rhizoctonia solani Associated with Black Scurf in Cyprus

During 2011, 96 sclerotial isolates of Rhizoctonia solani were collected from potato tubers from all main potato-cultivating regions of Cyprus. All isolates were found to be multinucleate. Characterization of anastomosis groups (AG) based on hyphal anastomosis reactions showed that 91 isolates belonged to AG3 and 5 to AG4. Sequence analysis of the internal transcribed spacer (ITS) regions (ITS1 and ITS2) of ribosomal DNA (rDNA) of 68 isolates confirmed the prevalence of AG3. In addition, phylogenetic analysis found that AG3 isolates were of the potato type, distinctly separated from the AG3 tobacco type, while AG4 isolates were separated into two different subgroups (HGI and HGII). Temperature studies showed that isolates belonging to both AG4 subgroups had significantly higher optimum growth temperatures compared with AG3. In vitro sensitivities to the fungicide pencycuron, in terms of concentrations where 50% growth inhibition was observed, ranged from 0.012 to 0.222 mu g/ml. Pathogenicity and aggressiveness of the isolates was determined on 'Annabelle' potato sprouts and seedlings of a number of selected hosts, based on crop rotations followed in Cyprus. The majority of the isolates were pathogenic to potato sprouts, with disease severity (DS) values ranging from 0 to 88%. Mean DS values were statistically different among AG and subgroups, with AG4-HGI (69.25%) and AG4-HGII (3.12%) being the most and least aggressive, respectively. However, AG4-HGII isolates were the most aggressive in all rotational hosts tested, while AG3 isolates were the least aggressive. More specifically, the highest DS levels by AG4-HGI were recorded to barley, by AG4-HGII to lettuce and melon, and by AG3 isolates to vetch. This is the first comprehensive study to elucidate the AG composition, pathogenicity and other biological aspects of R. solani isolates associated with potato black scurf in Cyprus

Optimizing efficacy of new postharvest fungicides and evaluation of sanitizing agents for managing citrus green mold

Three new fungicides, azoxystrobin, fludioxonil, and pyrimethanil, that belong to different chemical classes are highly effective in managing citrus green mold and are being registered for postharvest use in the United States. Recirculating in-line drenches provided a significantly improved efficacy compared with standard low-volume spray applications. To prevent pathogen contamination of drench solutions, two oxidizing disinfectants, sodium hypochlorite and hydrogen peroxide/peroxyacetic acid (HPPA) solutions, were evaluated. Inhibition of conidial germination of Penicillium digitatum was dependent on the pH of the solution and the exposure time for each sanitizing agent. Chlorine (50 mg/liter) and HPPA (2,700 mg/liter) effectively inhibited germination in 40- and 240-s exposures, respectively, at pH 7. All fungicides tested were compatible and effective with HPPA, whereas fludioxonil, azoxystrobin, and thiabendazole, but not imazalil and pyrimethanil, were compatible with chlorine. In laboratory studies, sodium bicarbonate (SBC, 3%) significantly increased the efficacy of the three fungicides (250 mg/liter) and had no adverse effect on their stability in aqueous solutions. Fludioxonil (300 mg/liter)-SBC mixtures were still highly effective when applied 24 h after fruit inoculation. In experimental packingline studies, SBC or SBC-chlorine improved the efficacy of fludioxonil, whereas azoxystrobin was effective with and without these additives. Heating of drench solutions of fludioxonil (300 mg/liter) to 50°C did not improve decay control. In conclusion, in-line recirculating drench applications and fungicide-sanitizer-SBC mixtures significantly increased fungicide efficacy and provide an integrated approach for optimizing fungicide efficacy. These strategies also should minimize the selection for resistant isolates of the pathoge