Novel hosts of the Eucalyptus canker pathogen Chrysoporthe cubensis and a new Chrysoporthe species from Colombia

The pathogen Chrysoporthe cubensis (formerly Cryphonectria cubensis) is best known for the important canker disease that it causes on Eucalyptus species. This fungus is also a pathogen of Syzygium aromaticum (clove), which is native to Indonesia, and like Eucalyptus, is a member of Myrtaceae. Furthermore, C. cubensis has been found on Miconia spp. native to South America and residing in Melastomataceae. Recent surveys have yielded C. cubensis isolates from new hosts, characterized in this study based on DNA sequences for the ITS and β-tubulin gene regions. These hosts include native Clidemia sericea and Rhynchanthera mexicana (Melastomataceae) in Mexico, and non-native Lagerstroemia indica (Pride of India, Lythraceae) in Cuba. Isolates from these hosts and areas group in the sub-clade of C. cubensis accommodating the South American collections of the fungus. This sub-clade also includes isolates recently collected from Eucalyptus in Cuba, which are used to epitypify C. cubensis. New host records from Southeast Asia include exotic Tibouchina urvilleana from Singapore and Thailand and native Melastoma malabathricum (Melastomataceae) in Sumatra, Indonesia. Consistent with their areas of occurrence isolates from the latter collections group in the Asian sub-clade of C. cubensis. DNA sequence comparisons of isolates from Tibouchina lepidota in Colombia revealed that they represent a new sub-clade within the greater Chrysoporthe clade. Isolates in this clade are described as Chrysoporthe inopina sp. nov., based on distinctive morphological differences.     

Microsatellite variation in the freshwater snail Physa acuta

We present data for seven new markers isolated from a microsatellite‐enriched DNA library from the freshwater snail Physa acuta. Six of them appear to be conserved in Physa cubensis. These loci might be useful for analysing population structure and reconstructing the story of invasion of freshwaters on a worldwide scale by P. acuta.     

Resurgence of cucurbit downy mildew in the United States: Insights from comparative genomic analysis of Pseudoperonospora cubensis

Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew (CDM), is known to exhibit host specialization. The virulence of different isolates of the pathogen can be classified into pathotypes based on their compatibility with a differential set composed of specific cucurbit host types. However, the genetic basis of host specialization within P. cubensis is not yet known. Total genomic DNA extracted from nine isolates of P. cubensis collected from 2008 to 2013 from diverse cucurbit host types (Cucumis sativus, C. melo var. reticulatus, Cucurbita maxima, C. moschata, C. pepo, and Citrullus lanatus) in the United States were subjected to whole‐genome sequencing. Comparative analysis of these nine genomes confirmed the presence of two distinct evolutionary lineages (lineages I and II) of P. cubensis. Many fixed polymorphisms separated lineage I comprising isolates from Cucurbita pepo, C. moschata, and Citrullus lanatus from lineage II comprising isolates from Cucumis spp. and Cucurbita maxima. Phenotypic characterization showed that lineage II isolates were of the A1 mating type and belonged to pathotypes 1 and 3 that were not known to be present in the United States prior to the resurgence of CDM in 2004. The association of lineage II isolates with the new pathotypes and a lack of genetic diversity among these isolates suggest that lineage II of P. cubensis is associated with the resurgence of CDM on cucumber in the United States.     

Distribution,Host Range and Disease Severity of Pseudoperonospora cubensis on Cucurbits in the Czech Republic

Cucurbit downy mildew, caused by Pseudoperonospora cubensis, is a major cucumber disease in the Czech Republic. Disease prevalence, host range and disease severity were evaluated from 2001 to 2009. The geographical distribution of P. cubensis was assessed on ca 80–100 locations per year in two main regions of the Czech Republic (central and southern Moravia, and eastern, northern and central Bohemia). Infection by P. cubensis was observed primarily on cucumber (Cucumis sativus) but only on the leaves. During the study, disease prevalence ranged from 66 to 100%. The majority of C. sativus crops were heavily infected at the end of the growing season (second half of August). Generally, P. cubensis was present at high or very high disease severity. The loss of foliage results in the reduction in the quality and quantity of marketable yield of fruit. Pseudoperonospora cubensis was widespread across the whole area of the Czech Republic studied. Very rarely, infection was recorded in muskmelon (Cucumis melo) and Cucurbita moschata. Of other pathogens, the most frequently recorded was the cucurbit powdery mildew (Golovinomyces cichoracearum and Podosphaera xanthii).     

Host specificity shapes population structure of pinworm parasites in Caribbean reptiles

Host specificity is one of the potential factors affecting parasite diversification because gene flow may be facilitated or constrained by the number of host species that a parasite can exploit. We test this hypothesis using a costructure approach, comparing two sympatric pinworm parasites that differ in host specificity – Parapharyngodon cubensis and Spauligodon anolis – on the Puerto Rican Bank and St. Croix in the Caribbean. Spauligodon anolis specializes on Anolis lizards, whereas P. cubensis parasitizes Anolis lizards as well as many other species of lizards and snakes. We collected lizards from across the Puerto Rican Bank and St. Croix, sampled them for S. anolis and P. cubensis and generated nuclear and mitochondrial sequence data from the parasites. We used these data to show that P. cubensis is comprised of multiple cryptic species that exhibit limited population structure relative to S. anolis, which is consistent with our prediction based on their host specificity. We also provide evidence that the distribution of P. cubensis species is maintained by competitive exclusion, and in contrast to previous theoretical work, the parasites with the greatest number of host species also reach the highest prevalence rates. Overall, our results are consistent with the hypothesis that host specificity shapes parasite diversification, and suggest that even moderate differences in host specificity may contribute to substantial differences in diversification.     

Distribution,habitat disturbance and pollination of the endangered orchid Broughtonia cubensis (Epidendrae: Laeliinae)

The geographical distribution, population structure and pollination ecology are key aspects in the conservation and management of rare orchids. Here, we address these aspects and the main threats affecting the endangered Cuban orchid Broughtonia cubensis. This rewardless orchid is self‐compatible, but pollinator dependent. However, seed production can be negatively affected by insect‐mediated selfing. Three species of small bee (genera Ceratina and Lasioglossum) act as pollinators. As in the case of other nectarless orchids, we detected two species of plant producing large amounts of nectar in the area, the floral morphology of which closely resembles that of B. cubensis. The simultaneous flowering of these species could positively affect the reproductive success of B. cubensis. Nonetheless, the fitness of this orchid in natural conditions is low, possibly related to strong pollen limitation. To the problems arising from reduced fitness is added the fact that its historical distribution range has been greatly reduced in recent years. Throughout this study, we have detected dramatic reductions in the population sizes, in some cases as a result of human plundering, but also as a consequence of hurricanes. Based on the results of this study, we propose some guidelines to manage and conserve this orchid. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 345–357.     

NO和钙信使系统在草酸诱导黄瓜叶片抗霜霉病中的作用

通过草酸及其与不同抑制剂亚甲基蓝、EGTA、氯丙嗪和Li+组合处理黄瓜叶片,研究了草酸与抑制剂不同处理组合方式对黄瓜叶片POD活性和叶片病情指数的影响,探讨NO、钙信使系统在草酸诱导叶片抗霜霉病中的作用.结果显示,10～70mmol/L草酸均能不同程度诱导黄瓜叶片POD活性的升高,提高叶片对黄瓜霜霉病的抗病性,降低叶片病情指数,并以30mmol/L效果最好.4种抑制剂分别与30mmol/L草酸同时或先于草酸处理,或草酸处理后一定时间再用抑制剂处理,均明显抑制黄瓜叶片POD活性的升高及病情指数的降低.研究表明,NO、Ca2+、钙调素(CaM)和磷酸肌醇均可能参与了草酸诱导黄瓜霜霉病抗性的信号转导过程.     

Spatio‐temporal Analysis of the Effects of Hurricane Ivan on Two Contrasting Epiphytic Orchid Species in Guanahacabibes,Cuba

Hurricanes represent the dominant type of disturbance in many tropical coastal forests. Here, we focus on mortality of epiphytic orchids caused by hurricane Ivan in the Guanahacabibes National Park (Cuba) and subsequent population recovery. We analyzed different aspects of hurricane damage on two contrasting epiphytic orchids, Broughtonia cubensis and Dendrophylax lindenii, as observed in three plots of coastal vegetation and in three plots of semi‐deciduous forest, respectively. First, we quantified the damage to host trees and orchids and explored if hurricane damage depended on height, size, or identity of the host tree. Second, we used mark connection and mark correlation functions to conduct a detailed analysis of small‐scale spatial patterns in hurricane damage for host trees and orchids. Finally, we analyzed the degree of recovery after Ivan during the 6 yr following the storm. Damage of B. cubensis host trees was independent of height and size, but Ivan severely affected larger and higher host trees of D. lindenii. Spatial analysis revealed non‐random structure in damage that differed between species. Broughtonia cubensis exhibited small‐scale spatial correlation in the proportion of damaged orchids, whereas D. lindenii did not. Dendrophylax lindenii showed ‘patchy’ damage patterns, correlated with height, but B. cubensis did not. The relative growth rate of B. cubensis for the 5–17 mo following Ivan was only moderately reduced and fully recovered in subsequent years, whereas that of D. lindenii was severely reduced the first year and did not fully recover thereafter. We hypothesize that differences in the host, vegetation type, and the traits of the two orchids contribute to the different responses to the hurricane.     

Polymorphic microsatellite markers for the ant Plagiolepis pygmaea

Detailed studies on kin structure, mating patterns and dispersal in social insects require highly polymorphic markers, of which the most commonly used today are DNA microsatellites. Here we characterize 10 polymorphic microsatellite markers for the ant Plagiolepis pygmaea. We also investigated the within‐genus applicability of the markers on P. xene, a social parasite of the source species. In addition, we tested amplification of the markers in three species of the genera Formica and Lasius. Eight of the markers also amplified in P. xene and were polymorphic. Seven markers amplified in at least one other formicine ant.     

Pathogenic variability of Pseudoperonospora cubensis in Gangetic Alluvial region of West Bengal,India

Cucurbits are tremendous economically important vegetable crops and extensively cultivated in tropical and subtropical part of the world during both rainy and winter seasons. Biotic stresses are of paramount importance in causing economic losses in cucurbits. Downy mildew, a foliar disease caused by an Oomycetes, Pseudoperonospora cubensis (Berk. & Curt.) Rostow, is one of the most destructive pathogens of cucurbits especially in wet and temperate regions, with worldwide distribution. Isolates of pointed gourd and ivy gourd showed no symptoms with most of the cucurbits hosts except pointed gourd and ivy gourd. P. cubensis isolate of pointed gourd differed from other isolates and may belong to different pathotype. The results of this investigation suggest that high variability of P. cubensis in terms of different pathotypes exists in the Gangetic Alluvial Region of West Bengal. Based on morphological characterisation, nine isolates of P. cubensis are grouped into four clusters by using hierarchical cluster analysis.     

Conspecificity of <Emphasis Type="Italic">Endothia eugeniae</Emphasis> and <Emphasis Type="Italic">Cryphonectria cubensis</Emphasis>: a re-evaluation based on morphology and DNA sequence data

Cryphonectria cubensis and Endothia eugeniae are fungal pathogens of Eucalyptus and clove that were reduced to synonymy on the basis of results of cross-inoculation studies, isozyme analysis, cultural studies, and morphology. A previous phylogenetic study on Cryphonectria, based on sequence variation in the ITS region of the ribosomal RNA operon, also supported the conspecificity of C. cubensis and E. eugeniae, but was based on only one E. eugeniae isolate. New collections from clove in Brazil and Indonesia have become available, providing the opportunity to reconsider the conspecificity of C. cubensis and E. eugeniae. The occurrence of C. cubensis on clove was confirmed based on morphological comparisons and phylogenetic analyses of ribosomal DNA and β-tubulin gene sequence data. In addition to C. cubensis, other fungi morphologically similar to Cryphonectria species on the basis of their orange stromata were present on some clove specimens, but no isolates were available for these fungi. Furthermore, some isolates, for which no herbarium material exists, grouped separately from the C. cubensis clade and closer to the Cryphonectria clade. The presence of more than one closely related fungus on clove raises questions relating to the legitimacy of the synonymy of E. eugeniae and C. cubensis. Based on the presence of C. cubensis on the type specimen of E. eugeniae, we recognize the synonymy of the two fungi but provide evidence that other fungi, more closely related to Cryphonectria spp. than to C. cubensis, are present on clove.     

Fungicide resistance in cucurbit downy mildew – methodological, biological and population aspects

Cucurbit downy mildew, caused by Pseudoperonospora cubensis, is among the most devastating diseases of cucurbitaceous plants. In spite of improved cultural practices and breeding for resistant cultivars, chemical control is still a very important tool to manage the disease. During the last several decades, many fungicides from various chemical classes have been developed. The occurrence of strains of P. cubensis resistant/tolerant to some fungicides encouraged research of this phenomenon. The first part of this article summarises the many different methodological approaches such as field trials, in vitro testing on active plant tissues or molecular diagnoses developed for the detection of resistant/tolerant strains of P. cubensis, as well as methods to collect and maintain pathogen isolates. The second part outlines the commonly used fungicides to control P. cubensis and their features like systemicity, biological and biochemical mode of action and translocation behaviour within plants. The last part deals with geographical aspects such as first appearance of resistance problems, distribution of resistance, temporal development of resistance under selection pressure by a fungicide, fitness of resistant subpopulations in competition with sensitive ones in the absence of a fungicide, as well as genetic and molecular sources of resistance.     

Analyzing the Spatial Structure of Broughtonia cubensis (Orchidaceae) Populations in the Dry Forests of Guanahacabibes,Cuba

A current key issue in ecology is the role of spatial effects on population and community dynamics. In this paper, we test several hypotheses related to spatial structures and coexistence of epiphytic tropical orchid species with special emphasis on the endemic species Broughtonia cubensis. More specifically, we explored the spatial structure of orchid–host plant communities at three different levels of organization (occupied vs. nonoccupied host trees, trees with B. cubensis vs. other orchids, and reproductive vs. nonreproductive B. cubensis plants). We mapped all potential host trees and orchids at three 20 × 20 m plots and applied techniques of spatial point pattern analysis such as mark connection and mark correlation functions to evaluate departures from randomized communities. We found spatial aggregation of trees with epiphytic orchids and segregation between trees with and without epiphytic orchids, and that there was an intraspecific spatial aggregation of B. cubensis in relation to the other seven epiphytic orchid species. Furthermore, we found spatial aggregation of reproductive B. cubensis individuals and segregation between reproductive and nonreproductive individuals on their phorophytes. Thus, orchid–host plant communities show hierarchical spatial structuring with aggregation and segregation at different levels of organization. Our results point to an enhancement of local species in the coexistence of tropical epiphytic orchid communities, by reducing competition through niche differentiation.     

Variation of Fungicide Resistance in Czech Populations of Pseudoperonospora cubensis

During the growing seasons between the years 2001 and 2004, 98 isolates of Pseudoperonospora cubensis from nine regions of Czech Republic were collected and screened for tolerance/resistance to the three frequently used fungicides (propamocarb, fosetyl‐Al, metalaxyl). Fungicides were tested in five different concentrations, using a floating disc bioassay. Fungicide effectiveness varied considerably. Propamocarb appeared most effective and all the isolates collected in the years 2001–2003 were found sensitive to all tested concentrations [607–9712 μg active ingredient (a.i.)/ml]. In 2004, some strains with increased resistance to propamocarb were detected. These strains were characterized by tolerance at the lowest concentrations (607 μg a.i./ml, eventually on 1214 μg a.i./ml); however, they were controlled by 2428 μg a.i./ml. Fosetyl‐Al was effective at the recommended concentration of 1600 μg a.i./ml against all isolates. However, the occurrence of isolates (collected in 2001) which sporulated at low concentrations (400 and 800 μg a.i./ml) indicated that the selection for tolerance occurs in the pathogen population. Nevertheless, this phenomenon was not confirmed with the P. cubensis isolates collected between the years 2002 and 2004. Metalaxyl was found ineffective, because 97% of the isolates showed the resistance to the recommended concentration (200 μg a.i./ml), and the other 3% of isolates expressed tolerant response. The majority of the isolates showed profuse and/or limited sporulation at higher concentrations (400 and 800 μg a.i./ml). A substantial shift to highly metalaxyl resistant strains was evident in the Czech P. cubensis populations during 2001–2004.     

Study of physio-biochemical responses elicited by potassium phosphite in downy mildew-infected cucumber plants

Potassium phosphite (KPhi) is widely used as a resistance inducer to protect plants against fungal pathogens. In the present study, the effect of KPhi on the activation of defence-related enzymes and biochemicals in Pseudoperonospora cubensis-challenged cucumber plants was investigated. Cucumber plants were treated with KPhi before or after inoculation with P. cubensis and leaf samples were collected at different time courses for physiological and biochemical assessments. Results revealed that the activity of reactive oxygen species (ROS)-scavenging enzymes like catalase, guaiacol peroxidase, superoxide dismutase and ascorbate peroxidase as well as proline and total carbohydrates contents were significantly increased by KPhi application, while hydrogen peroxide (H₂O₂) concentration, as a disease damage indicator was reduced. The maximum activity of ROS-scavenging system was achieved 3–4 days after KPhi application. These findings suggest that KPhi application prior to pathogen infection efficiently triggers plant defence responses which may reduce the disease severity.     

Assessment of SCAR markers to design real‐time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize

Aims: To assess the applicability of sequence characterized amplified region (SCAR) markers obtained from BOX, ERIC and RAPD fragments to design primers for real‐time PCR quantification of the phytostimulatory maize inoculants Azospirillum brasilense UAP‐154 and CFN‐535 in the rhizosphere. Methods and Results: Primers were designed based on strain‐specific SCAR markers and were screened for successful amplification of target strain and absence of cross‐reaction with other Azospirillum strains. The specificity of primers thus selected was verified under real‐time PCR conditions using genomic DNA from strain collection and DNA from rhizosphere samples. The detection limit was 60 fg DNA with pure cultures and 4 × 10³ (for UAP‐154) and 4 × 10⁴ CFU g^?1 (for CFN‐535) in the maize rhizosphere. Inoculant quantification was effective from 10⁴ to 10⁸ CFU g^?1 soil. Conclusion: BOX‐based SCAR markers were useful to find primers for strain‐specific real‐time PCR quantification of each A. brasilense inoculant in the maize rhizosphere. Significance and Impact of the Study: Effective root colonization is a prerequisite for successful Azospirillum phytostimulation, but cultivation‐independent monitoring methods were lacking. The real‐time PCR methods developed here will help understand the effect of environmental conditions on root colonization and phytostimulation by A. brasilense UAP‐154 and CFN‐535.     

QTL Mapping of Downy Mildew Resistance in an Introgression Line Derived from Interspecific Hybridization Between Cucumber and Cucumis hystrix

Downy mildew (DM), caused by Pseudoperonospora cubensis (Berk. & M.A. Curtis) Rostovzev, is a worldwide major disease of cucumbers (Cucumis sativus L.). By screening 10 introgression lines (ILs) derived from interspecific hybridization between cucumber and the wild Cucumis, C. hystrix, through a whole plant assay, one introgression line (IL52) was identified with high DM‐resistance. IL52 was further used as a resistant parent to make an F₂ population with ‘changchunmici’ (susceptible parent). The F₂ population (300 plants) was investigated for DM‐yellowing, DM‐necrosis and DM‐resistance in the adult stage. A genetic map spanning 642.5 cM with 104 markers was constructed and used for QTL analysis from the population. Three QTL regions were identified on chromosome 5 and chromosome 6. By interval mapping analysis, two QTLs for DM‐resistance were determined on chromosome 5 (DM_5.1 and DM_5.2), which explained 17.9% and 14.2% of the variation, respectively. QTLs for DM‐yellowing were in the same regions as DM‐resistance. For DM‐necrosis, by interval mapping analysis, one QTL was determined on chromosome 5 (Necr_5.1) that explained 18.3% of the variation and one on chromosome 6 (Necr_6.1) that explained 13.9% of the variation. Our results indicated that the identification of molecular markers linked to the QTLs could be further applied for marker‐assisted selection (MAS) of downy mildew resistance in cucumber.     

The role of potassium phosphite in chlorophyll fluorescence and photosynthetic parameters of downy mildew-challenged cucumber Cucumis sativus plants

The effect of potassium phosphite (KPhi) on the photosynthetic parameters of cucumber plants inoculated with Pseudoperonospora cubensis was investigated in the present study. Cucumber plants were treated with KPhi before or after inoculation with P. cubensis and leaf samples were collected at different time courses for assessments. Results showed that in pathogen-inoculated plants Fv/Fm was decreased up to 3%. The rate of quantum photosynthetic was also decreased significantly in inoculated plants. Downy mildew led to a decrease in chlorophyll amount which in turn reduced the efficiency of photosystem II. In the KPhi-treated leaves, chlorophyll a and b decreased by 72% and 68%, respectively. Remarkable reduction in the efficiency of photosystem II as well as increased lipid membrane disruption, led to increased lipid peroxidation rate of the membranes up to 52%. The results of this study indicate the mitigating role of potassium phosphite in reducing the adverse effects of pathogen and maintaining the photosynthetic apparatus efficiency in cucumber plants.