Molecular phylogenetic and morphological analyses of Oidium heveae, a powdery mildew of rubber tree

Powdery mildew of rubber tree caused by Oidium heveae is an important disease of rubber plantations worldwide. Identification and classification of this fungus is still uncertain because there is no authoritative report of its morphology and no record of its teleomorphic stage. In this study, we compared five specimens of the rubber powdery mildew fungus collected in Malaysia, Thailand, and Brazil based on morphological and molecular characteristics. Morphological results showed that the fungus on rubber tree belongs to Oidium subgen. Pseudoidium. Nucleotide sequence analysis of the ribosomal DNA internal transcribed spacer (ITS) region and the large subunit rRNA gene (28S rDNA) were conducted to determine the relationships of the rubber powdery mildew fungus and to link this anamorphic fungus with its allied teleomorph. The results showed that the rDNA sequences of the two specimens from Malaysia were identical to a specimen from Thailand, whereas they differed by three bases from the two Brazilian isolates: one nucleotide position in the ITS2 and two positions in the 28S sequences. The ITS sequences of the two Brazilian isolates were identical to sequences of Erysiphe sp. on Quercus phillyraeoides collected in Japan, although the 28S sequences differed at one base from sequences of this fungus. Phylogenetic trees of both rDNA regions constructed by the distance and parsimony methods showed that the rubber powdery mildew fungus grouped with Erysiphe sp. on Q. phillyraeoides with 100% bootstrap support. Comparisons of the anamorph of two isolates of Erysiphe sp. from Q. phillyraeoides with the rubber mildew did not reveal any obvious differences between the two powdery mildew taxa, which suggests that O. heveae may be an anamorph of Erysiphe sp. on Q. phillyraeoides. Cross-inoculation tests are required to substantiate this conclusion.     

Two Erysiphe species associated with recent outbreak of soybean powdery mildew: results of molecular phylogenetic analysis based on nuclear rDNA sequences

 Serious outbreaks of powdery mildew by a fungus belonging to the mitosporic genus Oidium subgenus Pseudoidium have been reported on soybean (Glycine max) in a wide area of eastern Asia since 1998. The taxonomic and phylogenetic placement of the causal fungus has not yet been determined because of lack of the perfect stage. We found ascomata having mycelioid appendages on a single leaf of soybean infested by powdery mildew. Molecular phylogenetic analysis was conducted based on a total of 14 sequences of the rDNA internal transcribed spacer (ITS) region from 13 soybean and wild soybean (Glycine soja) materials collected in Japan, Korea, Vietnam, and the United States, combined with 47 sequence data obtained from the DNA databases. It was revealed that two Erysiphe species were associated with the outbreak of soybean powdery mildew. There was 16% difference between the two species in genetic divergence of the ITS sequence. One species with perfect stage has an ITS sequence identical to that of Erysiphe glycines on Amphicarpaea and is identified as Erysiphe glycines based on the ITS sequence and morphology of ascomata. The second species, without the perfect stage, is likely to be Erysiphe diffusa (= Microsphaera diffusa), known as the fungus causing soybean powdery mildew in the United States, because the ITS sequences are identical to those from materials collected in the United States. However, we need materials having ascomata of E. diffusa to confirm the species name. Received: March 15, 2002 / Accepted: May 22, 2002     

Simultaneous modification of three homoeologs of TaEDR1 by genome editing enhances powdery mildew resistance in wheat

Wheat (Triticum aestivum L.) incurs significant yield losses from powdery mildew, a major fungal disease caused by Blumeria graminis f. sp. tritici (Bgt). enhanced disease resistance1 (EDR1) plays a negative role in the defense response against powdery mildew in Arabidopsis thaliana; however, the edr1 mutant does not show constitutively activated defense responses. This makes EDR1 an ideal target for approaches using new genome‐editing tools to improve resistance to powdery mildew. We cloned TaEDR1 from hexaploid wheat and found high similarity among the three homoeologs of EDR1. Knock‐down of TaEDR1 by virus‐induced gene silencing or RNA interference enhanced resistance to powdery mildew, indicating that TaEDR1 negatively regulates powdery mildew resistance in wheat. We used CRISPR/Cas9 technology to generate Taedr1 wheat plants by simultaneous modification of the three homoeologs of wheat EDR1. No off‐target mutations were detected in the Taedr1 mutant plants. The Taedr1 plants were resistant to powdery mildew and did not show mildew‐induced cell death. Our study represents the successful generation of a potentially valuable trait using genome‐editing technology in wheat and provides germplasm for disease resistance breeding.     

Artificial elevation of glutathione contents in salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) reduces susceptibility to the powdery mildew pathogen Euoidium longipes

• The effects of elevated glutathione levels on defence responses to powdery mildew (Euoidium longipes) were investigated in a salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) and wild‐type cv. Xanthi plants, where salicylic acid (SA) contents are normal.
• Aqueous solutions of reduced glutathione (GSH) and its synthetic precursor R‐2‐oxothiazolidine‐4‐carboxylic acid (OTC) were injected into leaves of tobacco plants 3 h before powdery mildew inoculation.
• SA‐deficient NahG tobacco was hyper‐susceptible to E. longipes, as judged by significantly more severe powdery mildew symptoms and enhanced pathogen accumulation. Strikingly, elevation of GSH levels in SA‐deficient NahG tobacco restored susceptibility to E. longipes to the extent seen in wild‐type plants (i.e. enhanced basal resistance). However, expression of the SA‐mediated pathogenesis‐related gene (NtPR‐1a) did not increase significantly in GSH or OTC‐pretreated and powdery mildew‐inoculated NahG tobacco, suggesting that the induction of this PR gene may not be directly involved in the defence responses induced by GSH.
• Our results demonstrate that artificial elevation of glutathione content can significantly reduce susceptibility to powdery mildew in SA‐deficient tobacco.

     

First Report of Frosty Mildew on Salix koreensis Caused by Mycopappus alni in Korea

A frosty mildew was observed on leaves of Salix koreensis in two localities of Korea during 2011 and 2012. The main signs and symptoms were expressed as conical white to cream coloured tufts of the causal fungus on the brown lesions, followed by premature defoliation. Based on morphological observations, cultural characteristics and phylogenetic analyses of rDNA‐ITS, the fungus was identified as Mycopappus alni, which has been known to be associated with frosty mildews on Alnus spp., Betula spp., Crataegus chlorosarca and Pyrus pyrifolia. Pathogenicity test was conducted twice with the same results, fulfilling Koch's postulates. This is the first case of Salix–Mycopappus association as well as the first report of frosty mildew on S. koreensis.     

Genetic dissection of a TIR‐NB‐LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine

The most economically important diseases of grapevine cultivation worldwide are caused by the fungal pathogen powdery mildew (Erysiphe necator syn. Uncinula necator) and the oomycete pathogen downy mildew (Plasmopara viticola). Currently, grapegrowers rely heavily on the use of agrochemicals to minimize the potentially devastating impact of these pathogens on grape yield and quality. The wild North American grapevine species Muscadinia rotundifolia was recognized as early as 1889 to be resistant to both powdery and downy mildew. We have now mapped resistance to these two mildew pathogens in M. rotundifolia to a single locus on chromosome 12 that contains a family of seven TIR‐NB‐LRR genes. We further demonstrate that two highly homologous (86% amino acid identity) members of this gene family confer strong resistance to these unrelated pathogens following genetic transformation into susceptible Vitis vinifera winegrape cultivars. These two genes, designated r esistance to P lasmopara v iticola (MrRPV1) are the first resistance genes to be cloned from a grapevine species. Both MrRUN1 and MrRPV1 were found to confer resistance to multiple powdery and downy mildew isolates from France, North America and Australia; however, a single powdery mildew isolate collected from the south‐eastern region of North America, to which M. rotundifolia is native, was capable of breaking MrRUN1‐mediated resistance. Comparisons of gene organization and coding sequences between M. rotundifolia and the cultivated grapevine V. vinifera at the MrRUN1/MrRPV1 locus revealed a high level of synteny, suggesting that the TIR‐NB‐LRR genes at this locus share a common ancestor.     

Molecular phylogenetic analyses reveal a close relationship between powdery mildew fungi on some tropical trees and Erysiphe alphitoides, an oak powdery mildew

To investigate the phylogenetic relationships among the powdery mildew fungi of some economically important tropical trees belonging to Oidium subgenus Pseudoidium, we conducted molecular phylogenetic analyses using 30 DNA sequences of the rDNA internal transcribed spacer (ITS) regions and 26 sequences of the domains D1 and D2 of the 28S rDNA obtained from the powdery mildews on Hevea brasiliensis (para rubber tree), Anacardium occidentale (cashew), Bixa orellana, Citrus spp., Mangifera indica (mango), and Acacia spp. The results indicate that the powdery mildew fungi isolated from these tropical trees are closely related to one another. These powdery mildews are also closely related to E. alphitoides (including Erysiphe sp. on Quercus phillyraeoides). Because of the obligate biotrophic nature of the powdery mildew fungi, the relationship between powdery mildews and their host plants is conservative. However, the present study suggests that a particular powdery mildew species has expanded its host ranges on a wide range of the tropical trees. This article also suggests that a powdery mildew fungus distributed in temperate regions of the Northern Hemisphere expanded its host ranges onto tropical plants and may be a good example of how geographical and host range expansion has occurred in the Erysiphales.     

First Report of Anthracnose of Ledebouriella seseloides Caused by Colletotrichum gloeosporioides in Korea

In 2012, dark brown spots were observed on leaves of Ledebouriella seseloides (Fang Feng) in several research plots located at the Goseong Agricultural Research Extension services in Gyeongam Province, Republic of Korea. A fungus was isolated from the infected plants which produced pink‐coloured spores in mucilage on PDA and conidial morphology suggested that the causal agent was Colletotrichum gloeosporioides. Internal transcribed spacer sequences of the pathogen showed 99% identity to those of C. gloeosporioides. Pathogenicity of the isolate was proved by Koch's postulates. This is the first report of anthracnose in L. seseloides caused by C. gloeosporioides.     

Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) as a potential mycoparasite on Sphaerotheca fuliginea (Ascomycotina: Erysiphales)

Hyphomycete Paecilomyces fumosoroseus that is well known as saprophytic and entomopatogenic fungus was investigated for its mycoparasitism on the cucumber powdery mildew pathogen. Mycoparasitism was documented by using standard bioassay and SEM. Effects of mycoparasitism were evaluated in three types of experiments. Paecilomyces fumosoroseus was applied in the form of graded suspensions into a colony of powdery mildew on a leaf segment. Interaction between both fungi was observed as the percentage of colonized area vs. experimental time. In the second experiment, young cucumber plants were sprayed with a suspension of Paecilomyces fumosoroseus 24 h before inoculation of Sphaerotheca fuliginea. Pre-treatment with P. fumosoroseus reduced development and spreading of powdery mildew infection significantly 15 days post-inoculation in contrast to pre-treatments with sulfur fungicide and distilled water. The development of pure culture powdery mildew under determined experimental conditions was observed and compared with treated variants. In the third experiment, mildewed plants were treated with a suspension of P. fumosoroseus. The control treatments with sulfur fungicide and distilled water were tested. Effects of P. fumosoroseus on the dispersion of powdery mildew during a 21-day period were observed.P. fumosoroseus suppressed the development and spread of cucumber powdery mildew significantly during the time of the experiment. The mechanical and physical damages and disruptions of vegetative and fruiting structures of powdery mildew were recorded under light microscopy and S.E.M.Results were concluded in pursuance to differences between the natural behaviour and development of S. fuliginea on cucumber plants treated with P. fumosoroseus and non-treated plants.     

Sooty Mould on Hibiscus rosa‐sinensis Caused by Leptoxyphium kurandae is Associated with Extrafloral Nectaries

In August 2013, sooty mould was observed on Chinese hibiscus (Hibiscus rosa‐sinensis) in a propagation nursery in Seoul, Korea. The sooty mould initially developed at the junction between the leaf blade and leaf petiole and then dispersed along the vein on the abaxial surface. The fungal growth pattern on the plants was quite different from general sooty moulds growing on honeydew secreted by insects on the plants. On the basis of the morphological characteristics and phylogenetic analysis using the internal transcribed spacer rDNA, this fungus was identified as Leptoxyphium kurandae. A pathogenicity test was carried out to fulfil Koch's postulates. Through field observation and a pathogenicity test, we found an association between the sooty mould and extrafloral nectaries. To our knowledge, this is the first report of sooty mould caused by L. kurandae on the extrafloral nectaries of H. rosa‐sinensis.     

The First Record and Characterization of Powdery Mildew (Erysiphe pachypodiae sp. nov.) on Pachypodium lamerei (Apocynaceae)

During the summer and autumn of 1999, symptoms of powdery mildew disease were first observed on Pachypodium lamerei in the Czech Republic. White lesions of irregular shape appeared on leaf margins and spread towards the central vein of the leaf, often followed by necrosis of leaf tissue. In the spring of 2000, the sexual stage (cleistothecia) also appeared on infected leaves. Based on the observations of the morphology of its anamorph and teleomorph stage as well as on results from inoculation experiments, the identity and origin of this powdery mildew species are discussed. Based on the pseudoidium anamorph, this fungus may be clearly assigned to Erysiphe emendation, probably to section Uncinula, since the cleistothecial appendages are non‐mycelioid and occassionally circinate at the apex. The fungus is described as a new species Erysiphe pachypodiae sp. nov.     

Pythium aphanidermatum Causing Pythium Rot on Lampranthus spectabilis in Korea

Pythium aphanidermatum was found to be associated with rotting of leaves and stems in Lampranthus spectabilis in the summers of 2013 and 2014. Infected plants were initially characterized by water‐soaked and discoloured tissue, which are soon covered with cottony aerial hyphae. Subsequently, infected tissues wilted, leaves and stems appeared desiccated, and infected plants died. Pathogenicity of a representative isolate was proved, fulfilling Koch's postulates. Sequence of the internal transcribed spacer region of ribosomal DNA from the isolate shared 100% identity with that of P. aphanidermatum. To our knowledge, this is the first report of P. aphanidermatum causing leaf and stem rot on L. spectabilis in Korea.     

Phylogeny and taxonomy of powdery mildew fungi of Erysiphe sect. Uncinula on Carpinus species

A phylogenetic analysis of the Erysiphe with uncinuloid ascoma appendages (Erysiphe section Uncinula, Erysiphales, Ascomycota) on Carpinus spp. was done using sequences of the rDNA ITS regions and the D1/D2 domains of the 28S rDNA. These results, combined with morphological data, revealed a complex consisting of several distinct taxa. These included the already described Erysiphe carpinicola on C. japonica distinguishable from the Erysiphe sp. on C. betulus and C. tschonoskii as well as the one on C. laxiflora. Thus, it was shown that Oidium carpini, described from Europe on Carpinus betulus, the powdery mildew with uncinula-like ascomata, recently found in Europe on this host, as well as an Erysiphe on C. tschonoskii in Japan, described previously as E. carpinicola, all belong to a single new species, named E. arcuata in this paper. As the powdery mildew on C. laxiflora was also distinct from other known species, it is named E. carpini-laxiflorae in this paper. The already described E. pseudocarpinicola and Erysiphe sp. on Carpinus cordata are two additional taxa, which are morphologically and genetically distinguished from the other species of Erysiphe sect. Uncinula on Carpinus spp.     

Two new hosts of anamorphic Erysiphe quercicola: Cinnamomum camphora and Murraya paniculata

Based on collections of powdery mildews (Erysiphales) in Taiwan and combined molecular and morphological analyses, camphor tree (Cinnamomum camphora) and orange jasmine (Murraya paniculata) are recognized as new hosts of the anamorph of the powdery mildew Erysiphe quercicola. The anamorphic powdery mildew on C. camphora has been known as Pseudoidium cinnamomi, but its relationship to a teleomorph was unknown. For M. paniculata as substrate of powdery mildew, only an anamorphic Cystotheca species has been named. Morphological investigation of the fungus on this host shows that the specimens from Taiwan belong to another genus because of the lack of fibrosin bodies. Analysis of internal transcribed spacer sequences indicates that the anamorphic powdery mildews on camphor and orange jasmine belong to a clade representing E. quercicola, with the teleomorph found only on oak species (Quercus, Fagaceae), but with its anamorph reported from a broad host range, particularly in the tropics.     

A novel strain of Pseudozyma aphidis from mulberry parasitises the conidia of mulberry powdery mildew fungus Phyllactinia sp. and its biocontrol effect in the fields

Plant surface is colonised with a vast community of non-pathogenic epiphytic microorganisms which play an important role in host defence. In the present study, we reported a fungus from mulberry leaf surface that showed an antagonistic effect against mulberry powdery mildew fungal pathogen Phyllactinia sp. This novel isolate is a yeast-like fungus that was identified as Pseudozyma aphidis CNm2012 based on morphologic and phylogenetic analysis. According to our research, P. aphidis CNm2012 directly acted on the powdery mildew conidia via parasitism which caused conidial atrophy, collapse and eventually, cleavage and death. During the parasitic process, we found the isolate could gather around the conidia of Phyllactinia sp. and form hyphae that grew on the conidial surface and utilise the conidia as a nutrient source. Field studies revealed that P. aphidis CNm2012 could suppress the disease incidence of mulberry powdery mildew caused by Phyllactinia sp., and reduce the disease severity. To our knowledge, it is the first report of P. aphidis directly against powdery mildew fungus Phyllactinia spp. by parasitism. Our results indicated that P. aphidis CNm2012 could be served as an environmentally friendly alternative of chemical pesticides to manage mulberry powdery mildew disease.     

Temporal Dynamics of Powdery Mildew (Oidium neolycopersici) and its Effects on the Host Growth Dynamics of Tomato

Controlled glasshouse experiments were conducted to investigate the temporal progress of powdery mildew and its effects on host dynamics of tomato, without and with one fungicide application. Healthy tomato transplants (5‐ to 6‐week old) were artificially inoculated with powdery mildew, and disease progress as well as host growth were monitored in both fungicide sprayed and unsprayed treatments and compared with non‐inoculated plants. Actual disease severity on a plant basis increased in unsprayed plants reaching maximum severity in the proportionate range of 0.53–0.83. One fungicide spray significantly reduced the maximum disease severity by two‐ to fourfolds. Despite adjustments for defoliation, declines in the proportion of disease severity between successive assessments were evident. Whereas the estimated growth rates of diseased plants were significantly lower than that of healthy plants, no significant differences were observed in the maximum leaf area formed of inoculated and non‐inoculated plants. A considerable effect of the powdery mildew epidemics was manifested through hastened shrivelling and defoliation of diseased leaves within the tomato canopy. An average of 18–29% and 40–52% of leaves had abscised from the plant canopy at the last date of assessment in sprayed and non‐sprayed plants, respectively. Accordingly, defoliation accounted for 14–33.3% and 58.3–63.1% losses in leaf area of sprayed and non‐sprayed plants, respectively. Duration of healthy leaf area and yield of inoculated plants were also significantly reduced by powdery mildew epidemics.     

The Lr34 adult plant rust resistance gene provides seedling resistance in durum wheat without senescence

The hexaploid wheat (Triticum aestivum) adult plant resistance gene, Lr34/Yr18/Sr57/Pm38/Ltn1, provides broad‐spectrum resistance to wheat leaf rust (Lr34), stripe rust (Yr18), stem rust (Sr57) and powdery mildew (Pm38) pathogens, and has remained effective in wheat crops for many decades. The partial resistance provided by this gene is only apparent in adult plants and not effective in field‐grown seedlings. Lr34 also causes leaf tip necrosis (Ltn1) in mature adult plant leaves when grown under field conditions. This D genome‐encoded bread wheat gene was transferred to tetraploid durum wheat (T. turgidum) cultivar Stewart by transformation. Transgenic durum lines were produced with elevated gene expression levels when compared with the endogenous hexaploid gene. Unlike nontransgenic hexaploid and durum control lines, these transgenic plants showed robust seedling resistance to pathogens causing wheat leaf rust, stripe rust and powdery mildew disease. The effectiveness of seedling resistance against each pathogen correlated with the level of transgene expression. No evidence of accelerated leaf necrosis or up‐regulation of senescence gene markers was apparent in these seedlings, suggesting senescence is not required for Lr34 resistance, although leaf tip necrosis occurred in mature plant flag leaves. Several abiotic stress‐response genes were up‐regulated in these seedlings in the absence of rust infection as previously observed in adult plant flag leaves of hexaploid wheat. Increasing day length significantly increased Lr34 seedling resistance. These data demonstrate that expression of a highly durable, broad‐spectrum adult plant resistance gene can be modified to provide seedling resistance in durum wheat.     

The inhibitory effects of rose powdery mildew infection on the oviposition behaviour and performance of beet armyworms

In this study, we investigated whether the oviposition behaviour and performance of the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), on the rose cultivar Rosa chinensis Jacq. (Rosaceae) were affected when the plants were infected by rose powdery mildew, Podosphaera pannosa (Wallr.: Fr.) de Bary (Erysiphales). The bioassays revealed that the moths significantly avoided ovipositing on mildew‐infected rose leaves when compared to healthy leaves. Pupal weights, emergence rates, and fecundity decreased when the caterpillars were fed mildewed rose leaves. Further laboratory bioassays aimed to elucidate the effects of two volatile headspace extracts (separately collected from healthy and mildewed rose plants) on the oviposition behaviour and performance of the moths. The moths clearly preferred to oviposit on healthy rose leaves that were not sprayed with additional volatiles rather than on healthy leaves sprayed with the volatile extracts from mildewed plants. The mean number of eggs laid on the former leaves was more than six times higher than that laid on the latter leaves. Olfactory bioassays demonstrated that ovipositing moths were significantly more attracted to volatiles emitted by healthy rose leaves than to those emitted by mildew‐infected leaves. Similar results were obtained when comparisons were made between the volatile extracts collected from healthy and mildewed rose plants. Thus, volatiles from mildew‐infected roses have a strong inhibitory effect against the moths. These results indicated that rose volatiles play a role in the oviposition behaviour of the moths, and that the volatiles induced by powdery mildew might be used for insect control.