Identification of a constitutive 45 kDa soluble protein associated with resistance to downy mildew in muskmelon (Cucumis melo L.), line PI 124111 F

Analysis by sodium dodecylsulphate-polyacrylamide gel electrophoresis of the soluble proteins extracted from muskmelon (Cucumis melo L.), inbred line PI 124111F (PI) with resistance to Pseudoperonospora cubensis (Berk, et Curt.) Rost, and two susceptible cvs, Hemed and AnanasYokneam revealed the presence of a unique 45 kDa protein (P45) in the resistant but not in the susceptible plants. Subcellular fractionation indicated that P45 is a cytoplasmic soluble protein. It was detected in leaves and cotyledons but not in stems or roots. F₁ hybrid plants (Hemed × PI) that displayed only a partial resistance against P. cubensis expressed P45 at an intermediate level, whereas plants of the inbred generations F₅, F₇ and F₁₀ that displayed a high degree of resistance, expressed P45 at a level similar to the parental PI. Back-cross progeny plants [(Hemed × PI) × Hemed] segregate 1:2:1 partially resistant: susceptible: highly susceptible to the disease. The partially resistant plants showed an intermediate level of P45 (similar to F₁) whereas the highly susceptible plants had no P45, thus indicating cosegregation of P45 with resistance. The resistance of PI to P. cubensis was found to decrease at a colonization temperature of 12 °C. ³⁵S-methionine in vivo protein labelling revealed a reduction in the intensity of the P45 band in plants incubated for 11 h at 12 °C. The application of P45 in breeding programs of muskmelon for downy mildew resistance and its possible involvement in resistance to P. cubensis are discussed.     

Cucurbit downy mildew (<Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis>)—biology,ecology, epidemiology,host-pathogen interaction and control

Cucurbit downy mildew, caused by the oomycete Pseudoperonospora cubensis, is a devastating, worldwide-distributed disease of cucurbit crops in the open field and under cover. This review provides recent data on the taxonomy, biology, ecology, host range, geographic distribution and epidemiology of P. cubensis. Special attention is given to host-pathogen interactions between P. cubensis and its economically-important cucurbit hosts (Cucumis sativus, C. melo, Cucurbita pepo, C. maxima, and Citrullus lanatus); pathogenic variability in P. cubensis at the species, genus, and population levels; and, differentiation of pathotypes and races. Genetics and variability of host resistance and cellular and molecular aspects of such resistance are considered. A focus is given to methods of crop protection, including prevention and agrotechnical aspects, breeding for resistance—classical and transgenic approaches, chemical control and fungicide resistance. Novel technologies in biological and integrated control are also discussed. This review also summarizes the most important topics for future research and international collaboration.     

Structure and temporal shifts in virulence of Pseudoperonospora cubensis populations in the Czech Republic

The structure and temporal dynamics of the virulence of Pseudoperonospora cubensis (causal agent of cucurbit downy mildew) were studied in pathogen populations in the Czech Republic from 2001 to 2010. A total of 398 P. cubensis isolates collected from Cucumis (Cm.) sativus, Cm. melo, Cucurbita (Cr.) maxima, Cr. pepo, Cr. moschata and Citrullus lanatus were analysed for variation in virulence (pathotypes). Virulence was evaluated on a differential set of 12 genotypes of cucurbitaceous plants. All isolates of P. cubensis were characterized by their level of virulence (classified according the number of virulence factors, VF; low VF = 1–4, medium VF = 5–8, high VF = 9–12): high (75%), medium (24%) and low (1%). The structure and dynamics of virulence in the pathogen populations were expressed by pathotypes using tetrad numerical codes and a total of 67 different pathotypes of P. cubensis were determined. The most susceptible group of differentials was Cucumis spp., while the lowest frequency of virulence was recorded on Cr. pepo ssp. pepo, Ci. lanatus and Luffa cylindrica. A high proportion (c. 90%) of isolates was able to infect cucurbit species Benincasa hispida and Lagenaria siceraria, which are not commonly cultivated in the Czech Republic or elsewhere in central Europe. In the recent pathogen populations (2008–2010) there was prevailing frequency (70–100%) of isolates with high numbers (9–12) of virulence factors. ‘Super pathotype’ 15.15.15 was often observed in the study within the pathogen populations and was one of the four most frequently recorded pathotypes. Pseudoperonospora cubensis populations shifted to a higher virulence over time. From 2009 the pathogen population changed dramatically and new pathotypes appeared able to establish natural and serious infection of Cucurbita spp. and Ci. lanatus, which was not observed in 2001–2008. Generally, virulence structure and dynamics of P. cubensis populations are extremely variable in the Czech Republic.     

Resistance mechanism to carboxylic acid amide fungicides in the cucurbit downy mildew pathogen Pseudoperonospora cubensis

BACKGROUND: Pseudoperonospora cubensis, the causal oomycete agent of cucurbit downy mildew, is responsible for enormous crop losses in many species of Cucurbitaceae, particularly in cucumber and melon. Disease control is mainly achieved by combinations of host resistance and fungicide applications. However, since 2004, resistance to downy mildew in cucumber has been overcome by the pathogen, thus driving farmers to rely only on fungicide spray applications, including carboxylic acid amide (CAA) fungicides. Recently, CAA‐resistant isolates of P. cubensis were recovered, but the underlying mechanism of resistance was not revealed. The purpose of the present study was to identify the molecular mechanism controlling resistance to CAAs in P. cubensis. RESULTS: The four CesA (cellulose synthase) genes responsible for cellulose biosynthesis in P. cubensis were characterised. Resistant strains showed a mutation in the CesA3 gene, at position 1105, leading to an amino acid exchange from glycine to valine or tryptophan. Cross‐resistance tests with different CAAs indicated that these mutations lead to resistance against all tested CAAs. CONCLUSION: Point mutations in the CesA3 gene of P. cubensis lead to CAA resistance. Accurate monitoring of these mutations among P. cubensis populations may improve/facilitate adequate recommendation/deployment of fungicides in the field. Copyright © 2011 Society of Chemical Industry     

Downy mildew of Cucurbits (Pseudoperonospora Cubensis): the Fungus and its hosts,distribution, epidemiology and control

Taxonomy of the genusPseudoperonospora, morphology ofPseudoperonospora cubensis (Berk, et Curt.) Rostow. and occurrence of its oospores, are described briefly. A list is presented of over 40 cucurbitaceous host species, representing about 20 genera, on whichP. cubensis has been recorded. Two or more races exist in Japan and the United States, but not in Europe or the Middle East. The distribution ofP. cubensis is widest on all continents on cucumbers (70 countries) and muskmelon (50 countries); onCucurbita and watermelons it extends to about 40 and 25 countries, respectively. P. cubensis may overwinter as oospores, though this seems rare, and on wild hosts or crops grown in the open or under cover. Airborne sporangia may also reach cooler countries from regions with mild winters. Apart from the leaf wetness essential for infection, the factors determining disease progress are: rate of foliage growth and physiological age of the host; amount of primary inoculum available, light, and the rate at which lesions necrotize. The interaction of these factors is described for early, mid-season, and late crops. Losses caused byP. cubensis depend on the growth stage at which the crop is attacked, and on the rate of foliage and pathogen development. Breeding has produced downy mildew resistant lines of cucumbers, used chiefly in the United States, and some resistant lines of melons and watermelons. The most important agricultural practices used to restrict downy mildew development are proper irrigation management and avoidance of sowing in proximity to infected crops. Success of control by protectant chemicals depends largely on proper timing of applications. Proximity of inoculum sources, hours of leaf wetness, age of crop, and irrigation practices are the principal factors that determine when to begin treatments. These factors and rate of leaf formation determine the frequency of applications. Application of systemic fungicides is much easier to time correctly.     

The significance of pronounced divergences in the distribution ofpseudoperonospora cubensis on its crop hosts

Pseudoperonospora cubensis is common throughout the world onCucumis crops (cucumber, melon); less common onCucurbita crops (squash, pumpkin, marrow), from which it is absent in Europe and parts of Asia; and more restricted onCitrullus, Luffu andLagenaria spp. This divergence in distribution is due chiefly to different physiological races in various countries. This must be taken into account in quarantine and breeding work. Resistance of some local cultivars, and the differential effects of environment on infection, add to the uneven distribution.     

Combined effects of salt stress and cucurbit downy mildew (Pseudoperospora cubensis Berk. and Curt. Rostov.) infection on growth,physiological traits and antioxidant activity in cucumber (Cucumis sativus L.) seedlings

Abiotic and biotic stresses both decrease quality and quantity of cultivated plants. We compared responses of Cucumis sativus L. cvs Sardes and Beith alpha which were treated with 50 or 100 mM NaCl and/or infected by Pseudoperospora cubensis Berk. and Curt. Rostov.. Both cultivars were affected remarkably when fungus infection was combined with salt stress however; cv Sardes was found out to be relatively tolerant with sustainable growth performance, better leaf water status and lower lipid peroxidation levels under this condition. This relative tolerance could be related to the fine tuning of the activities of the antioxidative enzymes and proline levels.     

Characterization of Pseudoperonospora cubensis isolates from Europe and Asia using ISSR and SRAP molecular markers

Downy mildew caused by Pseudoperonospora cubensis is a major disease of cucurbits worldwide. New genotypes of the pathogen have recently appeared in the USA, EU and Israel causing breakdown of genetic resistance, expansion of host range, and the appearance of a new A2 mating type. Seventy-eight P. cubensis isolates were collected during 1996–2011 from cucurbits fields in different regions of Turkey, Israel and the Czech Republic and genetic diversity was analysed using highly polymorphic ISSR and SRAP molecular markers. The data acquired showed remarkable genetic diversity within and among the isolates. While isolates from Turkey and Czech Republic exhibited uniform genetic background, the isolates from Israel were clearly distinguished from the others. The results may indicate on migration and/or frequent sexual reproduction of the pathogen in Israel. Moreover the selected markers can be suggested for monitoring genetic diversity within P. cubensis isolates in further studies.     

Evaluation of Malus germplasm resistance to marssonina apple blotch

Marssonina apple blotch, caused by Diplocarpon mali (anamorph: Marssonina coronaria), is a serious disease of apple (Malus x domestica Borkh.). We evaluated the resistance of 28 cultivars and 39 species or biotypes after inoculating leaves with conidial suspensions in vitro and in vivo. Five accessions of the 28 apple cultivars showed a good level of resistance, while 16 displayed intermediate type. In contrast, resistance was good for eight accessions of species or biotypes and moderate for 19 accessions. Among these 67 candidates, more than 19 % had resistance against Marssonina apple blotch, indicating that resistant resources are abundant in that germplasm. These high-performing accessions can make valuable contributions to breeding programs for resistance against this disease.     

Increased susceptibility toPseudoperonospora cubensis in virus-infectedCucurbita pepo genotypes

Pseudoperonospora cubensis developed more and larger lesions, infected greater leaf areas and produced more sporangia on virus-infected (Vi) than on virus-free (Vf) leaves ofCucurbita pepo genotypes.Pseudoperonospora cubensis from Vi and Vf leaves did not vary in sporangial dimensions (length x width) or pathogenicity to different cucurbits. The preferential development and sporulation ofP. cubensis in Vi than in Vf leaves was due to increased host susceptibility.     

Identification of <Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis> using real-time PCR and high resolution melting (HRM) analysis

To develop a molecular method to identify Pseudoperonospora cubensis in cucumber leaves with signs of downy mildew, we compared the nucleotide sequences of ribosomal DNA-internal transcribed spacer, cytochrome oxidase II, and β-tubulin genes of P. cubensis and P. humuli isolates. Seven single nucleotide polymorphisms (SNPs) distinguished P. cubensis and P. humuli based on variations in β-tubulin sequences, and one specific primer set was designed for further analysis. Real-time PCR and high resolution melting analysis showed that the primer set can be used to specifically identify P. cubensis in cucumber leaves with downy mildew.     

Occurrence and molecular characterization of azoxystrobin resistance in cucumber downy mildew in Shandong province of China

Cucumber downy mildew caused byPseudoperonospora cubensis (Berk. and Curt.) Rostov. limits crop production in Shandong Province of China. Since management of downy mildew is strongly dependent on fungicides, a rational design of control programs requires a good understanding of the fungicide resistance phenomenon in field populations of the pathogen. A total of 106 and 97 isolates ofP. cubensis were obtained in 2006 and 2007, respectively. The EC₅₀ values for the growth of all the 106 isolates collected in 2006 were 0.0063–0.0688μg ml⁻¹ (average: 0.0196±0.0048μg ml⁻¹) azoxystrobin and these were therefore considered sensitive isolates. However, 57 field isolates ofP. cubensis of the 97 collected in 2007 with EC₅₀ values that ranged from 0.609 to >51.2μg ml⁻¹ were considered resistant to azoxystrobin. Fragments of the fungicide-targeted mitochondrial cytochromeb gene from total pathogen DNA were amplified using polymerase chain reaction and their sequences analyzed to elucidate the molecular mechanism of resistance. A single point mutation (GGT to GCT) in the cytochromeb gene, resulting in substitution of glycine by alanine at position 143, was found in the three selected azoxystrobin-resistant isolates of downy mildew. This substitution in cytochromeb exhibited different resistance levels, with the resistance factor from 21.15 to greater than 2618.9. In addition, the different resistance levels seemed to appear within 1 year (between 2006 and 2007). Therefore, growers of Shandong Province in China now are faced with a challenge in managing the azoxystrobin resistance in cucumber downy mildew. http://www.phytoparasitica.org posting March 10, 2008.     

河北省和山东省黄瓜霜霉病菌对氟吡菌胺的抗性及常规药剂对黄瓜霜霉病的田间防效

为明确黄瓜霜霉病菌Pseudoperonospora cubensis对氟吡菌胺的抗性时空动态,2011—2016年从河北省和山东省黄瓜主产区采集1 821株霜霉病菌,采用叶盘漂浮法检测供试菌株对氟吡菌胺的敏感性,并以茎叶喷雾法评估5种常规药剂对黄瓜霜霉病的田间防效。结果表明,河北省和山东省黄瓜主产区的黄瓜霜霉病菌对氟吡菌胺已普遍产生了低度抗性,平均抗性倍数为5.86;所有供试菌株的抗性频率为51.89%,其中低抗菌株及中抗菌株分别占36.18%和15.71%;平均抗性指数为0.34,每年检测的抗性频率、抗性倍数及抗性指数呈增长趋势,且随着监测区域的变化而变化。在河北省定兴县和山东省寿光市进行的田间防效试验结果显示,按推荐剂量喷施,687.5 g/L氟吡菌胺·霜霉威盐酸盐SC对黄瓜霜霉病防效显著高于常规对照药剂68%精甲霜灵·代森锰锌WG、58%甲霜灵·代森锰锌WP、80%代森锰锌WP及250 g/L嘧菌酯SC的防效,但防效明显下降,由2011年的92.58%～93.31%降至2016年的80.07%～80.82%。表明需要制定和实施抗药性治理对策,如限制山东和河北2省黄瓜的每个生长季节中687.5 g/L氟菌·霜霉威SC的使用不超过2次,而且要与不同作用机理的卵菌杀菌剂交替使用防治黄瓜霜霉病。     

Evaluation of world castor (Ricinus communis L.) germplasm for resistance to Fusarium wilt (Fusarium oxysporum f. sp. ricini)

Castor (Ricinus communis L.) is an important industrial oilseed crop grown worldwide. Wilt caused by Fusarium oxysporum f. sp. ricini is a devastating disease of this crop. The objective of this research was to identify stable sources of wilt resistance among the global castor germplasm collections available in India for use in cultivar improvement. The global collections comprising 1,779 Indian and 190 exotic accessions from 36 countries were screened against wilt in wilt sick plots at two sites in India in preliminary screening. None of the accessions showed high resistance to wilt, 133 accessions comprising 111 Indian and 22 exotic accessions representing 13 countries exhibited resistance. Thirteen of the 133 resistant accessions were tested further for multiple years in wilt sick plots and glasshouse under controlled artificial inoculation at two sites. All the 13 accessions consistently showed wilt resistance in both wilt sick plot and glasshouse at both sites in multiple years. Eleven of these 13 accessions were from India and two were from former USSR. Evaluation for agro-morphological traits identified high seed yielding and early maturing resistant accessions. Diversity analyses precisely revealed diversity among the resistant accessions. These 13 resistant accessions would be great value as donors of resistance.     

Conidial sporulation of <Emphasis Type="Italic">Stemphylium solani</Emphasis> under laboratory conditions and infectivity of the inoculum produced <Emphasis Type="Italic">in vitro</Emphasis>

Potato virus Y (PVY) is the type-species of the genus Potyvirus, family Potyviridae, being reported as a major tomato (Solanum lycopersicum L.) pathogen in several regions of the world. Pepper yellow mosaic virus (PepYMV) was originally described as a resistance-breaking Potato virus Y (PVY) isolate on Capsicum annuum L. cultivars, and afterwards it was also reported infecting tomatoes in Brazil. In the present work, a search for sources of resistance to both PepYMV and PVY was conducted in a collection of 119 accessions belonging to seven Solanum (section Lycopersicon) species. This germplasm was initially evaluated to PepYMV reaction by mechanical inoculation followed by symptom observations and ELISA. Potential PepYMV resistance sources were identified for the first time in S. habrochaites, S. peruvianum, S. corneliomuelleri, S. chilense, S. pimpinellifolium, and one accession derived from an interspecific cross (S. lycopersicum x S. peruvianum). A sub-group of 24 accessions with negative serology for PepYMV was also challenged with a PVY isolate, followed by serological and molecular detection with universal primers. Solanum habrochaites ‘L.03683’ and ‘L.03684’ were the only accessions found with stable resistance to both viruses. These results confirm S. habrochaites as the most important source of multiple resistance factor(s) to distinct Potyvirus species.     

2,2,2-三氟乙基苯并[1,2,3]噻二唑-7-甲酸酯对黄瓜霜霉病的诱导抗病性

为明确2,2,2-三氟乙基苯并[1,2,3]噻二唑-7-甲酸酯(简称TBTC)对蔬菜病害的诱导抗病效果,以黄瓜霜霉病Pseudoperonospora cubensis为研究对象,进行了该化合物在盆栽及田间小区条件下的诱导抗病试验。结果表明:TBTC对黄瓜霜霉病具有较好的诱抗活性,其中,盆栽试验中250 mg/L处理的平均防效为77.50%±1.79%,对照诱导抗病剂苯并噻二唑(BTH)的平均防效是71.72%±2.58%, 对照杀菌剂50%烯酰吗啉可湿性粉剂在有效成分500 mg/L处理下的平均防效为71.46%±3.01%;两年的田间试验结果显示,TBTC 250 mg/L的处理对黄瓜霜霉病的最高防效达83.94%±2.09%,而BTH的最高防效为72.39%±2.91%,50%烯酰吗啉可湿性粉剂250 mg/L的最高防效是89.08%±1.95%,表明TBTC对田间黄瓜的霜霉病可产生诱导抗病效果。     

Phylogenetic investigations in the downy mildew genus <Emphasis Type="Italic">Bremia</Emphasis> reveal several distinct lineages and a species with a presumably exceptional wide host range

Bremia lactucae is one of the most devastating and widespread pathogens in lettuce production worldwide. Despite its economical importance, uncertainty prevails about the species delimitation in the genus Bremia. Commonly, Bremia is considered to be monotypic, containing only Bremia lactucae, while taxonomists have described additional species, and molecular phylogenetic studies have shown significant sequence divergence between accessions from different hosts. Here, we report that several previously described species are genetically highly distinct from Bremia lactucae parasitic to Lactuca sativa. These include Bremia lapsanae, Bremia sonchicola, and Bremia taraxaci. In addition to these host-specific species, a plurivorous species is revealed, which infects hosts from three different tribes in the Asteraceae subfamilies Asteroideae and Carduoideae. The broad host range of clade 1 is exceptional for downy mildews and only paralleled by Pseudoperonospora cubensis, which infects a broad range of Cucurbitaceae. The taxonomic status of Bremia cirsii and of Bremia centaureae remains unresolved, as the accessions from Cirsium and Centaurea, respectively, did not form a monophylum but were partly contained in the plurivorous clade 1. Bremia lactucae was found to be restricted to Lactuca sativa and Lactuca serriola. Thus, it can be assumed that Bremia infections on weeds apart from Lactuca species do not pose a significant risk for lettuce production. However, it is unlikely that breeding resistance genes from Lactuca serriola into Lactuca sativa will result in durable resistance of lettuce to downy mildew disease, because the current study provides additional evidence that Bremia accessions from both hosts form a population continuum.     

Quantitation of resistance of cucumbers and cantaloups toPseudoperonospora Cubensis

Of 14 varieties and lines of cucumber and seven of cantaloup tested for resistance againstPseudoperonospora cubensis, only the cucumbers Poinsett and Chipper were found to be highly resistant. Growth chamber studies showed that resistance of young as well as of old (> 8-leaf-stage) plants was stable under considerably high (300 sporangia/cm²) inoculum loads. Resistance was conferred by both restriction of fungal growth and prevention of sporangial formation.     

Resistance to acylalanines in Pseudoperonospora cubensis1

Resistance to acylalanine fungicides in Pseudoperonospora cubensis, the downy mildew of cucurbits, is reviewed. Insensitive strains of the fungus predominated during the second year of metalaxyl application in cucumber glasshouses in Greece and Israel. Resistance was detected either on detached cucumber leaves floated on metalaxyl solutions or on potted plants treated (sprayed or drenched) with this fungicide. Metalaxyl-resistant strains of P. cubensis showed good pathogenicity and fitness, and competed favorably with the sensitive strains in the absence of the fungicide. A synergism between these two biotypes resulted in an increased virulence of the resistant forms on metalaxyl-treated plants. Metalaxyl-resistant strains of the pathogen exhibited cross resistance to other acylalanine fungicides. Strains resistant to acylalanines and to the chemically unrelated oomycete-fungicides fosetyl-Al and propamocarb were isolated in Israel, as well. In Greece, however, protective sprays with fosetyl-Al, cymoxanil and mancozeb gave good control of cucumber downy mildew when the pathogen was resistant to metalaxyl. P. cubensis is the first case of fungus which developed resistance to acylalanines in the field.     

Coincidence of virulence shifts and population genetic changes of Pseudoperonospora cubensis in the Czech Republic

Pseudoperonospora cubensis is an oomycete pathogen causing downy mildew disease on a variety of Cucurbitaceae, and has recently re‐emerged as a destructive disease on crops in this family, mainly on cucumber and squash. Multilocus sequence analysis (MLSA) of four mitochondrial and two nuclear DNA regions was used to detect changes in the genetic structure of P. cubensis populations occurring in the Czech Republic that might be associated with recently reported shifts in virulence. The analysed sample set contains 67 P. cubensis isolates collected from 1995 to 2012 in the Czech Republic and some other European countries. Sequence analyses revealed differences and changes in the genetic backgrounds of P. cubensis isolates. While all isolates sampled before 2009 exhibited the genotype of the subspecies of Clade II and were collected from cucumber, all samples collected from other hosts belonged to Clade I (P. cubensis sensu stricto) or were sampled from 2009 onwards. In addition, 67·16% of all post‐2009 isolates from Clade II had two heterozygous positions in their nrITS sequence, which suggests sexual reproduction and/or a mutational origin. Thus, the results indicate that, apart from the rise in prevalence of Clade I, the change in the genetic structure of P. cubensis populations may be linked with a hybridization or, less likely, a mutation event that rendered strains able to infect a broader spectrum of host species.