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

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

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

Exploring the potential of kresoxim-methyl as a priming agent towards protection of Medicago truncatula plants grown under abiotic stress conditions

Kresoxim-methyl (KM) belongs to the strobilurins, one of the most important classes of agricultural fungicides, displaying a direct effect on physiological and developmental plant processes thus leading to greening of plants via induction of hormonal biosynthesis. The aim of this study was to examine the effect of KM pre-treatment on Medicago truncatula plants (ecotype Jemalong A17) subjected to drought and salinity conditions, evaluating key physiological, biochemical and molecular parameters. Foliar application with 10-8M KM resulted in increased stomatal conductance and chlorophyll fluorescence in leaves compared with stressed plants under both stress conditions, while maximum values for both parameters were obtained following control treatment with 10-8M KM but no subsequent stress imposition. The protective role of KM was further supported by attenuation of cellular damage indicated by lipid peroxidation and ROS content measurements following KM pre-treatment under both stress conditions. However, while KM pre-treatment resulted in lowering of NO content compared with drought-stressed plants, no significant alterations were observed in NO content under salt stress. Furthermore, molecular analysis of antioxidant gene expression (AOX, GST, cAPX, FeSOD) by real-time qRT-PCR revealed differential regulation under both drought and salinity conditions for all the genes examined. KM pre-treatment under salinity conditions resulted in massive induction of all genes compared with stressed samples, whereas suppression of expression levels was observed under drought conditions indicating activation or suppression of the antioxidant pathway following KM pre-treatment, respectively. Moreover, data from microarray analysis using Affymetrix technology indicated a large number of genes belonging to several families that appear to be regulated in response to KM pre-treatment in comparison with respective stressed samples