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.     

Mycopappus quercus sp. nov., causing frosty mildew inQuercus acutissima

The causal fungus of frosty mildew inQuercus acutissima was taxonomically studied. Brown spots with conical white to yellowish tufts of the causal, fungus developed on the leaves, followed by successive defoliation. The fungus was regarded as a new species ofMycopappus, M. quercus, from the morphology of the multicellular propagules. The ascogenous stage and true conidia of the fungus were not observed, although sclerotia were formed on the fallen diseased leaves.     

Mycosphaerella chaenomelis sp. nov.: The teleomorph ofCercosporella sp., the causal fungus of frosty mildew inChaenomeles sinensis, and its role as the primary infection source

A leaf spot disease called frosty mildew was observed onChaenomeles sinensis throughout Japan. Small brown spots with white tufts occurred followed by successive defoliation. On the fallen leaves, minute black dots are formed. The causal fungus was regarded as a new species ofMycosphaerella, M. chaenomelis, andCercosporella chaenomelis in anamorph. Pathogenicity of the fungus was confirmed only inC. sinensis by inoculation experiments. Colonies of the fungus grew well on potato sucrose agar, and grew at 10–30°C with an optimum temperature of 25°C. The fungus overwintered on the fallen diseased leaves in the form of pseudothecia, and ascospores served as the primary infection source from April to June.     

Mycena auricoma,a new species ofMycena sectionRadiatae from Japan,andMycena spinosissima,a new record in Japan

Two lignicolous species ofMycena (Agaricales, Basidiomycetes) are described and illustrated from eastern, Japan:Mycena auricoma sp. nov., forming ephemeral coprinoid basidiomata and belonging to sectionRadiatae, was found on a dead fallen twig ofQuercus serrata. It appears to close to a Malaysian species,“Trogia” crinipelliformis. Mycena spinosissima in sectionSacchariferae, new to Japan, was collected from dead bark ofAphananthe aspera, a dead fallen inflorescence ofCryptomeria japonica, and a dead fallen twig ofQuercus serrata.     

Phylogeny and taxonomy of the oak powdery mildew Erysiphe alphitoides sensu lato

Phylogenetic analyses of Erysiphe alphitoides s. lat. using sequences of the rDNA ITS region and the D1/D2 domains of the 28S rDNA revealed a complex consisting of several genetically and morphologically distinguished taxa, including the already described Erysiphe alphitoides s. str. and E. hypophylla. The ascomata (chasmothecia) of E. hypophylla are morphologically very similar to those of E. alphitoides, but the two species are easily distinguishable by their symptoms, as well as the shape and size of the conidia. The fungus on Quercus phillyraeoides, distributed in warmer regions in southern Japan, is genetically clearly separated from E. alphitoides s. str., and morphologically characterized by having chasmothecia with appendages consistently shorter than the chasmothecial diameter. This fungus, named Erysiphe quercicola in this paper, is also able to infect some other oak species, and it is genetically identical with anamorphs on some tropical trees of other host genera. Collections of E. alphitoides s. lat. on Quercus acutissima and Q. variabilis, both belonging to Quercus sect. Cerris, are genetically distinct from E. alphitoides s. str., E. hypophylla and E. quercicola. They form two genetically and morphologically differentiated groups. The hypophyllous taxon on Q. acutissima and Q. variabilis, named Erysiphe hypogena in this paper, is characterized by forming distinctive persistent hypophyllous mycelial patches, causing necrotic discolouration of the host tissue. The epiphyllous taxon on these hosts, for which the name E. epigena is proposed, differs in having epiphyllous mycelium, smaller chasmothecia with fewer appendages, and does not cause leaf discolouration.     

Lecanosticta acicola, causal fungus of brown spot needle blight inPinus thunbergii, new to Japan

A needle blight disease with brown spots was found on ornamental trees ofPinus thunbergii in Shimane Prefecture, Japan. The causal fungus was identified asLecanosticta acicola, known as causal fungus of brown spot needle blight of pines recorded in the Americas, Europe, and China.Pinus thunbergii was heavily infected following inoculation with the fungus in June, but only slightly infected following inoculation in September. The mycelia of the fungus were raised and produced conidial masses on potato-dextrose and Waksman agars. They grew well at 20–25°C within the range of 5 to 35°C.     

Didymella fagi sp. nov. and its anamorphAscochyta fagi, causing the yellow leaf spot disease ofFagus crenata andQuercus mongolica var.grosseserrata in Japan

AnAscochyta fungus was obtained during a survey of leaf spot diseases ofFagus crenata in the mountains of Aomori prefecture in 1995. The pathogenicity of the fungus toF. crenata was confirmed by inoculation. Its teleomorph was first found on artificially infected leaves after the leaves were placed in an incubator at 5°C with a 12-h photoperiod (approximately 500lx, daylight strip lamps) for 4 mo. The fungus was found to be the causal agent of yellow leaf spots ofF. crenata andQuercus mongolica var.grosseserrata in the field. Comparison with similar fungi so far described from Fagaceae indicated that the anamorph isAscochyta fagi and the teleomorph is an undescribed species ofDidymella. The nameDidymella fagi is introduced for the teleomorph. Correlation between the two morphs has been proved by cultural, morphological and pathological studies.     

Brown spot ofRheum officinale in indonesia with special reference to the variation in conidia of the causal fungus

Brown spot disease ofRheum officinale, a traditional medicinal plant in South East Asia, was newly recorded from the highland area of West Java Province. After confirmation of its pathogenicity towardRheum officinale and comparison of its morphology with hitherto known species, the casual fungus was identified asAscochyta rhei. It produces widely varied conidia from unicellular and bacillar conidia (Phoma-type), 1-septate oblong conidia (Ascochyta-type) to 2–3-septate cylindric conidia. These conidia were sometimes formed within the same pycnidium. They could germinate easily and produce the similar colonies to each other. Supported by “Joint Study Project for Strengthening Research on Diseases of Industrial Crops in Indonesia, Japan International Cooperation Agency (JICA)” in BALITTRO.     

Stromatinia cryptomeriae sp. nov., the teleomorph ofGloeosporidina cryptomeriae causing twig blight of Japanese cedar

A new species,Stromatinia cryptomeriae, is described based on a specimen collected in Iwate Prefecture, Japan. It was found on fallen dead branches and twigs of Japanese cedar,Cryptomeria japonica. The morphology of isolates on potato-dextrose agar (PDA), which were obtained from single ascospores ofS. cryptomeriae, was identical withGloeosporidina cryptomeriae, the causal fungus of Japanese cedar twig blight. In an inoculation test using single ascospore isolates, many minute black spots (sclerotioid bodies; sclerotules) and acervuli ofG. cryptomeriae were formed on the necrotic lesions, which developed into typical symptoms of Japanese cedar twig blight. These results show thatStromatinia cryptomeriae is the teleomorph ofG. cryptomeriae. On PDA, the fungus grew over a range of about 1 to 25°C, with the optimum growth at about 15–20°C.     

A new species ofPestalosphaeria, the teleomorph ofPestalotiopsis neglecta

Three specimens of a newPestalosphaeria species were obtained from a twig ofQuercus myrsinaefolia from Kumamoto Pref., and from leaves ofRhododendron hybridum andRicinus communis from Izu Peninsula, Shizuoka Pref. The collected materials were kept moist for 1 to 2 mo after collection. Monoascospore isolates from each specimen produced identical colonies with black slimy masses of conidia on them. Morphological characteristics of the conidia accorded well with those ofPestalotiopsis neglecta not only hitherto recorded but also formed on the same specimen. Hence,Pestalosphaeria gubae sp. nov. is proposed for the new species, as the teleomorph ofPestalotiopsis neglecta.     

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.     

Redheadia quercus gen. et sp. nov., the teleomorph of Mycopappus quercus, the frosty mildew fungus in Quercus acutissima

The teleomorph of Mycopappus quercus causing frosty mildew in Quercus acutissima is described as a new genus and species, Redheadia quercus, in the Sclerotiniaceae. Apothecia sprout from sclerotia on the fallen infected leaves kept for 10 months at 5°C and subsequent incubation at 15°C under diffused room light. Typical zonate lesions and multicellular propagules of M. quercus are produced on Q. acutissima, by mycelial inoculation using an isolate from a single ascospore, confirming the teleomorph–anamorphic connection. No significant differences are observed between cultured colonies of isolates from the ascospore and those from the propagule. Sclerotia and microconidia of the fungus are produced on culture media.     

Growth-promoting factors for yeast cells ofParacoccidioides brasiliensis

Low-density seedings of yeast cells ofParacoccidioides brasiliensis give poor growth (as assessed by plating efficiency test) on conventional mycological agar media, and therefore growth-promoting factors for this fungus were sought. Water-extracts of yeast cells of sixP. brasiliensis isolates were all considerably effective in promoting the growth of low-density seedings ofP. brasiliensis isolates Pb-18 and Hachisuga, but had little effect on isolate Bt-4. Horse serum, at a concentration range of 2–4%, moderately or considerably promoted the growth of theseP. brasiliensis isolates. Combinations of the fungus cell extracts with horse serum were highly effective in promoting the growth of all of the fungal isolates. The fungus cell extracts showed siderophore (microbial iron carrier) activity. An iron-chelator, ethylenediaminetetraacetic acid, at a concentration of 100 μM also highly promoted the growth of the fungal isolates in the presence of horse serum, and ferric ion added to culture medium was considerably effective in the growth promotion. These results suggest that deficient utilization of external iron by the fungus cell is one of the growth-limiting processes for low-density seedings of yeast cells ofP. brasiliensis on conventional mycological agar media.     

Fusarium solani invader of the eggs of the insectPanstrongylus geniculatus in a vivarium

A strain ofFusarium solani sensu Snyder & Hansen invaded the eggs of the insectPanstrongylus geniculatus in a vivarium. None of the invaded eggs hatched. To establish experimentally the pathogenicity of thisFusarium species against the eggs ofP. geniculatus, the fungus and the eggs were incubated together under different relative humidities and temperatures. At 64% relative humidity and 26 °C, the fungus grew well colonizing and penetrating all of the chorions.Three embryos died and were also colonized byF. solani. Only 4 nymphs hatched and survived to day 20. It is concluded that the isolate ofF. solani was capable of colonizing and invading the chorion of the eggs under certain humidity and temperature conditions and cause the death of the embryos.     

Electrophoretic protein patterns of three species ofPhytophthora associated with black pod disease of cocoa (Theobroma cacao L.)

When electrophoretic profiles of native proteins from vegetative mycelia ofPhytophthora palmivora, Phytophthora capsici and Phytophthora citrophthora causing black pod disease of cocoa in India were compared on a single Polyacrylamide gel, the isolates of same species were readily distinguished both qualitatively by visual similarity in banding patterns and quantitatively by calculating similarity coefficients. Similarity coefficients were generally much higher between isolates within a species than between isolates of different species. The dendrograms obtained after unweighted pair grouping with arithmetic averaging cluster analysis, revealed that all the isolates ofPhytophthora capsici were highly homogenous and formed a single cluster. The isolates ofPhytophthora citrophthora were resolved into two electrophoretic types which were clustered into two distinct sub groups.Phytophthora palmivora formed a separate group. Thus, the results reveal that polyacrylamide gel electrophoresis can be used successfully in distinguishing species and sub groups within a species ofPhytophthora encountered on cocoa. CPCRl contribution No. 914.     

Growth analysis ofQuercus serrata seedlings withinMiscanthus sinensis grass canopies differing in light availability

The effects of different light regimes on the survival, growth and morphology ofQuercus serrata seedlings were studied in canopies ofMiscanthus sinensis. The seedlings of various ages (0–3 yr) were grown in three light regimes: under a denseM. sinensis canopy (TG plot) receiving 2.5%–8.7% of full sunlight, under a relatively sparse canopy (SG plot) receiving 3.8%–16.1% of light and in an adjacent open site (NG plot). There was a little difference in the survival ofQ. serrata seedlings among the three plots. Height and diameter of stem and total leaf area of the seedlings were significantly lower in the shadier plots. However, the first (bottom) flush of the stem was significantly longer in the TG plot than in the NG and SG plots. Total dry weights of individual 1- and 2-yr-oldQ. serrata seedlings in the TG plot were reduced to about one-twelfth of those in the NG plot. Although the relative proportion in dry weight of each organ did not differ significantly among the plots, leaf area ratio, specific leaf area and stem height per unit dry weight were significantly higher in shadier plots. The leaf area per unit stem height was increased considerably in the sunnier plots.     

Geosiphon pyriforme,a fungus forming endocytobiosis withNostoc (Cyanobacteria), is an ancestral member of the glomales: Evidence by SSU rRNA Analysis

Geosiphon pyriforme inhabiting the surface of humid soils represents the only known example of endocytobiosis between a fungus (Zygomycotina; macrosymbiont) and cyanobacteria (Nostoc; endosymbiont). In order to elucidate the taxonomical and evolutionary relationship ofGeosiphon pyriforme to fungi forming arbuscular mycorrhiza (AM fungi), the small-subunit (SSU) ribosomal RNA genes ofGeosiphon pyriforme andGlomus versiforme (Glomales; a typical AM fungus) were analyzed and aligned with SSU rRNA sequences of several Basidiomycetes, Ascomycetes, Chytridiomycetes, and Zygomycetes, together with all AM-fungal (Glomales) sequences published yet. The distinct group of the order Glomales, which includesGeosiphon, does not form a clade with any other group of Zygomycetes. Within the Glomales, two main lineages exist. One includes the families Gigasporaceae and Acaulosporaceae; the other one is represented by the genusGlomus, the members of which are very divergent.Glomus etunicatum andGeosiphon pyriforme both form independent lineages ancestral to the Glomales. The data provided by the present paper confirm clearly thatGeosiphon represents a fungus belonging to the Glomales. The question remains still open as to whether or notGeosiphon is to be placed within or outside the genusGlomus, since this genus is probably polyphyletic and not well defined yet.Geosiphon shows the ability of aGlomus-like fungus to form a “primitive” symbiosis with a unicellular photcautotrophic organism, in this case a cyanobacterium, leading to the conclusion that a hypothetical association of aGlomus-like fungus with a green alga as a step during the evolution of the land plants appears probable. Correspondence to: H. Gehrig     

Fusarium fractiflexum sp. nov. and two other species within theGibberella fujikuroi species complex recently discovered in Japan that form aerial conidia in false heads

Morphological and molecular phylogenetic analyses were conducted on 12 strains ofFusarium, deposited in MAFF asF. subglutinans (≡F. moniliforme var.subglutinans≡F. sacchari var.subglutinans) orFusarium sp. because they formed aerial conidia in false heads in the dark. These strains were resolved as three distinct species within theGibberella fujikuroi species complex. A new species,F. fractiflexum, and two species new to Japan,F. circinatum andF. concentricum, are described and illustrated and their morphological features are discussed.Fusarium fractiflexum, isolated from diseased yellow leaf spots ofCymbidium spp., is differentiated from other fusaria based on its yellowish colonies and aerial conidia formed in false heads in the dark and in zigzag-like conidial chains under black light. Japanese strains ofF. circinatum also formed elongate, coiled sterile hyphae. Phialidic aerial conidia with a pointed apex and a wedgeshaped base were found inF. concentricum cultured under black light and represent a new diagnostic character of the species, in addition to colonies with alternating concentric rings when cultured on PDA. Based on DNA sequences of the β-tubulin gene and two other loci, strains ofF. fractiflexum were resolved phylogenetically as members of the Asian clade of theG. fujikuroi species complex. In addition, Japanese strains ofF. circinatum andF. concentricum were phylogenetically identical to the ex-type strains.     

Specific endonuclease activity of integrase encoded by the mdg4 (gypsy) retrotransposon

To study the mechanism of precise excision ofgypsy from genomic sites, the integrase domain ofgypsy pol was cloned and expressed inEscherichia coli. The endonuclease activity of recombinant integrase was assayed with synthetic substrates corresponding to 3′-U5 ofgypsy LTR and to the known genomic insertion sites ofgypsy. Integrase nicked the 5′-A ⇓ YR-3′ triplet in the (+) strand of the double-stranded substrates; cleavage of a single-stranded substrate was nonspecific. Cleavage proved to be affected by the local conformation of the substrate: the (+) strand was cleaved more efficiently when the (−) strand had an unpaired base in the triplet and was not cleaved when the (−) strand was interrupted or branched. The triplet corresponded to the consensus region ofgypsy insertion (5′-YRYR ⇓ YR-3′), the site of cleavagein vitro coinciding with the site of insertionin vivo. The unique mechanism ofgypsy excision was assumed to depend to a great extent on the enzymic properties of its integrase.