Analysis Of ITS Sequences Research Articles

A radical shift in the taxonomy of Lepraria s.l.: Molecular and morphological studies shed new light on the evolution of asexuality and lichen growth form diversification

A combination of molecular phylogenetic analyses of ITS and mtSSU sequences, morphological and chemical analyses were used to investigate the lineages nominally included in the sterile lichen genus Lepraria. A core group (Lepraria s. str.) was resolved as sister to Stereocaulon. Species producing the secondary compounds argopsin, pannarin and usnic acid were found to belong to other lineages of lichen-forming ascomycetes. Study of Leprocaulon revealed that all species, except the type, likely represent members of Lepraria s. str. that have evolved a fruticose growth form. The correct name for the type species of Leprocaulon is shown to be L. quisquiliare, not L. microscopicum, and the genus is redefined to include several species previously placed in Lepraria. Leprocaulon quisquiliare is also shown to comprise two morphologically convergent species. The name is lectotypified and epitypified on material from the type region (Germany) and its application restricted to Old World populations. New World populations of L. quisquiliare are described as L. americanum. Leprocaulon, in its revised sense, is recognized in a new family (Leprocaulaceae) and order (Leprocaulales) sister to the Caliciales and including the genus Halecania. A new genus of Pilocarpaceae, Nelsenium, is introduced to accommodate Lepraria usnica. The status of Lepraria ecorticata is discussed in the context of usnic acid-producing Lecanora species. These nomenclatural novelties are proposed: (i) transfers from Leprocaulon to Lepraria: Lepraria albicans comb. nov., L. arbuscula comb. nov., L. congestum comb. nov., L. gracilescens comb. nov., L. pseudoarbuscula comb. nov., L. subalbicans comb. nov., L. tenellum comb. nov.; (ii) transfers from Lepraria to Leprocaulon: Leprocaulon adhaerens comb. nov., L. coriense, L. santamonicae comb. nov., L. terricola comb. nov. and L. textum comb. nov.; (iii) new taxa: Leprocaulales ord. nov., Leprocaulaceae fam. nov., Nelsenium gen. nov., Leprocaulon americanum sp. nov. and L. knudsenii sp. nov.

Discrimination of the common macroalgae (Ulva and Blidingia) in coastal waters of Yellow Sea, northern China, based on restriction fragment-length polymorphism (RFLP) analysis

Since 2007, reoccurring large-scale green algae blooms have caused deleterious effects to the estuarine ecosystem of Yellow Sea, northern China and subsequent economical losses. Previous surveys indicated the green tides were initiated in the coastal water of southern Jiangsu province where Porphyra farming was intensively conducted; however, the main ‘seed source’ of floating green algae is still under debate. Ulva prolifera was confirmed to be the major causative species of green tides. The multiple sympatric ulvoid species in the natural environment has complicated species identification in both field surveys and laboratory studies due to their morphological plasticity. Thus, we developed a genetic identification key based on restriction fragment length polymorphism (RFLP) analysis of the ITS nuclear marker to discriminate the common Ulva and Blidingia species in the Yellow Sea. Ten genetic lineages (1 in Blidingia, 9 in Ulva) were detected along the coast of China through phylogenetic analysis of ITS sequences. They can be separated by virtual restriction digestion using the four selected restriction enzymes (BspT107 I, EcoO109 I, Hin1 I and VpaK11B I). With additional PCR amplification of the 5S spacer region, we were able to discriminate U. prolifera from Ulva linza. Using this genetic key, we screened macroalgal samples collected from the coast of the Yellow Sea, and the results indicated 6 common lineages (U. prolifera, U. linza, Ulva compressa, Ulva pertusa, Clade 6 and Blidingia sp.) in this region, which could be explicitly distinguished by a single enzyme (BspT107 I) coupled with 5S spacer polymorphism. U. prolifera was confirmed to be present on the Porphyra aquaculture rafts with seasonal variation in the species composition. This genetic key will facilitate our long-term field surveys to investigate the origin of the floating U. prolifera and furthermore to explore its bloom dynamics along the coast of the Yellow Sea. It also provided a framework for the future inclusion of more Ulva species, which will expand the usage of this key.

Identification of pathogen of Aquilaria sinensis dieback disease

The research focused on identification the pathogen of Aquilaria sinensis dieback disease and confirmation the function of inducing agarwood formation by fungi. The morphological observation, rDNA ITS sequence analyses, Koch's postulates and pathogenicity test were used to identify the isolates. The isolates of the causal agent was Lasiodiplodia theobromae, which induced the plant to produce highly valuable agarwood. The first report of dieback disease of Aquilaria sinensis caused by L. theobromae, and our research results laid a theoretical foundation for agarwood production by using fungus.

Isolation and identification of pathogen of seedling blight of Platycodon grandiflorum

To isolate and identify pathogen of the seedling blight occurred in Platycodon grandiflorum. The morphological observation, rDNA ITS sequence analysis, and Koch's postulates were used to identify the isolates of the causal agent. The isolates of the causal agent was Rhizoctonia solani. The result confirmed that R. solani is the pathogen of seedling blight of P. grandiflorum.

Population genetic structure and conservation of the Azorean tree Prunus azorica (Rosaceae)

Prunus azorica is an endangered tree endemic to the Azores Archipelago, considered as a top priority species for conservation. Although propagation measures have been studied in detail, and a broad phylogeographic study on P. lusitanica was recently published, a detailed population genetics study devoted to Azorean taxon was lacking. To determine extant patterns of population genetic structure in P. azorica, we analysed eight populations from the five Azorean islands where the species presently occurs and the only extant individual from Flores Island. We also included samples of P. lusitanica subsp. hixa from the Canary Islands and Madeira, and of P. lusitanica subsp. lusitanica from mainland Portugal. Genotyping was undertaken for eight nuclear microsatellite polymorphic loci specifically isolated for P. azorica. Accessions of the different geographic regions were used to sequence ITS and trnL DNA regions. Regarding SSRs, the number of alleles ranged from 5 to 37 (mean = 12.6) per locus and from 2 to 64 per population (mean = 24). Our analysis showed a clear separation between samples from the Azores and those from other regions. Overall, Sao Miguel populations seemed to encompass the majority of the variability found within the archipelago. Regarding the Azorean populations only, the highest percentage of genetic variation was found within populations (92 %). Still, about 7 % of the variation was found among populations within islands. Expected heterozygosity ranged from values near 0 in the most depauperate populations up to 0.18. With a few exceptions, the level of differentiation between Azorean populations was generally low and gene flow was clearly above 1. Analysis of ITS sequences also detected differences between the Azores and the remaining regions but the trnL region did not reveal any variation. The genetic identity of P. azorica was recognised and thus should be preserved; however, the present results suggest that the Azorean taxon should be reinstated at the subspecies level.

Molecular analysis confirms that Botryodontia millavensis and Oxyporus philadelphi are conspecific

The aim of the present study is to elucidate if Oxyporus philadelphi (Parmasto) Ryvarden and Botryodontia millavensis (Bourdot & Galzin) Duhem & H. Michel are conspecific, as suggested by their similar morphology. The analysis of ribosomal DNA ITS sequences indicate that the specimens of O. philadelphi and B. millavensis belong to a single species. The species is closely related to the type species (B. cirrata) of the genus Botryodontia, and therefore the name B. millavensis should currently be used for this taxon. Botryodontia millavensis grows on a wide selection of host species—Juniperus communis, Philadelphus coronarius, Picea abies, Fraxinus excelsior, Lonicera sp., J. turcomanica—and is distributed widely in temperate Eurasia.

자연산 다묵장어, Lethenterone reissneri에서 발생한 물곰팡이병 원인체의 동정

자연산 다묵장어, Lethenterone reissneri에서 발생한 물곰팡이병의 원인체를 형태학적 및 유전학적 연구를 통하여 동정하였다. 분리균주는 격벽이 없는 균사를 형성하였고, 가늘고 긴 모양의 유주자낭 내에 활발한 운동성을 보이는 유주자가 여러 줄로 배열되었다가 유주자낭의 선단에서 휴면하지 않고 한꺼번에 방출되는 특징을 보였다. 한편 유성생식기관인 조란기와 조정기는 관찰되지 않았다. 또한 분리균주의 ITS sequence를 분석하고 Saprolegnia 속의 다른 분리균주와 유전학적 상관관계를 조사한 결과, S. parasitica가 속해있는 clade I에 분류되는 것을 확인할 수 있었다. 무성생식기관의 형태, 유주자 방출방식 및 ITS sequence의 phylogenetic analysis에 근거하여 자연산 다묵장어에서 관찰된 물곰팡이병의 원인체는 S. parasitica인 것으로 동정되었다. Saprolegnia isolate from wild brook lamprey was identified on the basis of its morphological and molecular characteristics. The isolates showed aseptic hyphae and clavate zoosporagium. Zoospores discharge was typically saprolegnoid. Neither oogomia nor antheridia was observed in this study. ITS sequence obtained from the isolate was compared with other Saprolegnia spp. to analyse their phylogenetic relationships. Results showed that the isolate belongs to clade I including Saprolegnia parasitica. Based on the asexual organs, zoospore discharge manner and ITS sequence analysis, the isolate was identified as S. parasitica.

Gangrenous Cutaneous Mucormycosis Caused by Rhizopus oryzae: A Case Report and Review of Primary Cutaneous Mucormycosis in China Over Past 20 Years

Cutaneous mucormycosis is a rare opportunistic infection caused by zygomycetes that can be rapidly fatal if unrecognized. We describe the clinical, histopathological, fungal and molecular features of a case of gangrenous cutaneous mucormycosis. The patient presented with great necrosis on his right forearm at the site of detained intravenous cannula needle. He had type II diabetes and chronic renal insufficiency. KOH mount of black eschar showed many broad, aseptate fungal hyphae with right-angle branching. PAS staining of the tissue sample revealed similar broad hyphae in the dermis and cutis. Fungal culture and ITS sequence analysis identified this fungus as Rhizopus oryzae. As no organ involvement was detected, the patient was diagnosed with primary cutaneous mucormycosis. Considering the poor state of the patient, complete excision of the infectious tissue was performed without skin graft instead of amputation. At the same time, intravenous liposomal amphotericin B was given, starting from a small dosage and increased to a total dosage amount of 5.45 g. The wound recovered well with granulation. We emphasize that early recognition and prompt therapy including the control of the primary diseases were important. In this article, we also reviewed the features of primary cutaneous mucormycosis reported in China over the last 20 years.

<i>Primulina mabaensis</i> (Gesneriaceae), a new species from a limestone cave of northern Guangdong, China

We describe and illustrate the new species Primulina mabaensis from northern Guangdong, China. Its generic placement in the recently recircumscribed Primulina is confirmed by phylogenetic analyses of ITS and trnL-F intron-spacer DNA sequences. Primulina mabaensis is similar to P. repanda var. guilinensis, differing from the latter by its much larger flowers with corollas 2.5–3.0 cm (vs. 8–10 mm) long and corolla tubes 2.0–2.5 cm (vs. 6–8 mm) long and ca. 7 mm (vs. ca. 3 mm) in diameter at the corolla mouth. Primulina mabaensis is currently only known from limestone caves and rock surfaces of a karst tower in the Maba Man Archeology Site.

Fungal endophyte diversity and community patterns in healthy and yellowing leaves of Citrus limon

Abstract Fungal endophytes on citrus plants have been little studied, and the effects of citrus diseases on their incidence and diversity have not been addressed. In this study, we examined the foliar fungal endophytes of Citrus limon in the vicinity of Yaounde, Cameroon, with emphasis on the differences between endophyte communities in healthy and yellowing leaves. From 82.3 % of the 480 leaf fragments, a total of 482 isolates were recovered and analysis of ITS sequences revealed 20 phylotypes. All fungal endophytes were ascomycetes and, except for one species, were common plant pathogens. Mycosphaerella and its anamorphs (34.2 % of all isolates), and Colletotrichum gloeosporioides (50.4 % of all isolates), were isolated most frequently. Mycosphaerellaceous species dominated in healthy leaves, and were absent from yellowing leaves. C. gloeosporioides was isolated significantly more frequently from yellowing than healthy leaves. Yellowing leaves had a significantly higher overall infection frequency but, in contrast, the least species diversity. Difference in the endophyte assemblages of healthy and yellowing leaves suggests that yellowing of leaves may facilitate the incidence of certain endophytes and impose growth inhibition on others.

Molecular Characterization of the Asian Tiger Mosquito Aedes albopictus (Skuse) (Diptera: Culicidae) in Northern Italy

Polymorphism of the gene COI encoding cytochrome C oxidase subunit I and of the rRNA second internal transcribed spacer (ITS2) was measured in Aedes albopictus collected in three locations in Piedmont region and one location in Liguria region in Italy. According to our data, besides the middle part of the COI gene usually used for analysis, there are also SNPs in the 5′ half of the gene, which can provide additional information about the polymorphism of A. albopictus populations. Based on COI gene, three various mitochondrial haplotypes were identified in A. albopictus from four north Italian sampling sites. Different sources of introductions can be considered. The variability between COI sequences of the north Italian A. albopictus is 0.001 while the variability between them based on the pairwise analysis of ITS2 sequences is 10 time more—0.011. For the first time, a 4% divergence between the populations of A. albopictus of tropical and subtropical origins was found on the basis of analyzing the ITS2. A relatively high level of variability of ITS2 between tropical and subtropical populations can be used to develop new markers for phylogenetic studies. No individuals with mitochondrial and nuclear haplotypes specific to tropical populations were found in the four north Italian sampling sites.

Polyporus submelanopus sp. nov. (Polyporales, Basidiomycota) from Northwest China

Polyporus submelanopus, collected from Qinghai Province, Northwest China, is described and illustrated based on both morphological and phylogenetic analyses. It is characterized by a terrestrial habit, black-stipitate basidiocarps, circular to angular pores, two kinds of generative hyphae bearing simple septa and clamp connections respectively, and cylindrical smooth hyaline basidiospores. Morphologically, P. submelanopus belongs to the melanopus group and resembles P. melanopus in sharing similar pilei with a shallow central depression, circular to angular pores, basidiospores, and a terrestrial habit. However, P. melanopus has smaller pores, thicker and entire dissepiment, fusoid cystidioles, and clamped generative hyphae only. The differences between P. submelanopus and P. badius, P. xinjiangensis, and P. tubaeformis are discussed as well. Phylogenetic ITS sequence analysis supports P. submelanopus within the melanopus group but distinctly separate from other sampled species of this group.

Tuber sinoaestivum sp. nov., an edible truffle from southwestern China

A new Tuber species, T. sinoaestivum, is described from southwestern China based on morphological and molecular analyses. The new species, which was found fruiting in association with Pinus armandii, has been previously misidentified as T. aestivum (=T. uncinatum). Although morphologically quite similar, T. sinoaestivum differs from the European species by more globose ascospores ornamented with a shallower reticulum. ITS sequence analyses show only 92% similarity between the two species, further supporting the taxonomic separation of T. sinoaestivum from T. aestivum.

Tuber sinosphaerosporum sp. nov. from China

A new white truffle species from China is described and illustrated. Tuber sinosphaerosporum is characterized by its white ascomata and globose ascospores ornamented with large reticulum meshes. The new species, supported by ITS sequence analysis, offers commercial value due to its moderate to relatively large size and possibly high yield in China.

New Insights into the Phylogeny of <I>Angelica</I> and its Allies (Apiaceae) with Emphasis on East Asian Species, Inferred from nrDNA, cpDNA, and Morphological Evidence

Classification of Angelica and its allies is complex and controversial, with previous phylogenetic studies restricted to examina- tion of small numbers of taxa using only nrDNA ITS sequences. In this study, phylogenetic analyses of nrDNA ITS and ETS sequences, cpDNA sequences (rps16 intron, rps16-trnK, rpl32-trnL, and trnL-trnT), and morphological data, supplemented by observations of fruit anatomy and micromorphology, were used to ascertain evolutionary relationships and confirm generic boundaries within Angelica s.l. (including Angelica, Archangelica, Coelopleurum, Czernaevia, and Ostericum), with emphasis on its East Asian members. Most species of Angelica s.l. fall into two major, disparate clades, the Ostericum clade and the Angelica group, with the latter comprising five major lineages that are distinguished both molecularly and morphologically: Angelica s.s. (including Czernaevia), Archangelica, Coelopleurum, Glehnia, and a newly identified Littoral Angelica clade. A North American Angelica clade was also distinguished in the ITS trees. Taxonomic realignments will be required, as many species of Angelica fall outside of Angelica s.s. and four species of Angelica occur outside of the Angelica group (A. hirsutiflora, A. oncosepala, A. paeoniifolia, and A. sinensis). The Angelica s.s. clade contains predominantly East Asian species and comprises five major lineages, two of which represent plants exclusively from the eastern Himalayas; each of these five lineages can also be defined morphologi- cally. The results obtained are significantly different from traditional treatments of Angelica s.l. and provide new insights into the phylogeny and classification of the group.

Diversity analysis of Beauveria bassiana isolated from infected silkworm in southwest China based on molecular data and morphological features of colony

Beauveria bassiana is an important entomopathogenic fungus that not only often causes infection and epidemics of wild insects but some strains also show pathogenicity to the silkworm, Bombyx mori. The present study is about diversity of B. bassiana isolated from the silkworm in southwest China. Five strains of B. bassiana were isolated from infected silkworm. Two isolates, GXtr1009 and GXtr1010, were isolated from infected silkworms treated with two kinds of biological pesticides applied in Guangxi province, and three isolates, SCsk1006, YNsk1106 and GXsk1011, were collected from naturally infected silkworms from different geographical locations in Yunnan and Sichuan. All of the isolates showed highly similar conidia and conidial fructification, but the colony characteristics demonstrated great differences among the isolates. The ITS and 18S rDNA sequence analysis was sufficient to identify all five isolates as B. bassiana. However, the dendrogram, based on the ISSR data, produced two large genetic groups. GXtr1009 and GXtr1010 comprised one group, and SCsk1006, YNsk1106 and GXsk1011 converged in a different large group. The results suggested that, although all of these five B. bassiana strains were pathogenic to silkworms, strains of biological pesticides could be differentiated from strains of naturally infected silkworm via ISSR analysis.

COLLETOTRICHUM: HOST SPECIFICITY AND PATHOGENICITY ON SELECTED TROPICAL AND SUBTROPICAL CROPS

Colletotrichum and its teleomorph Glomerella are considered major fungal plant pathogens worldwide. They cause significant economic damage to fruit crops in tropical, subtropical and temperate regions. Several Colletotrichum species or biotypes are known to cause disease in a single host such as C. acutatum and C. gloeosporioides on apple, avocado, mango, papaya, passiflora, strawberry and tamarillo. It is also common to find a single species infecting multiple hosts such as C. gloeosporioides on apple, avocado, mango, papaya, peach, strawberry, and other hosts. Cross-infection potential was shown for two species, C. gloeosporioides from limonium and C. acutatum from strawberry, when inoculating peach, pear, mango, nectarine and strawberry. Molecular analyses including species-specific PCR amplification and ITS sequencing was reliable for identification of Colletotrichum isolates infecting apple, avocado, mango, papaya, passiflora, peach, strawberry and tamarillo fruits. Sub-populations within C. acutatum, C. gloeosporioides and C. boninense were characterized using ITS sequence analysis, while the latter appears to be a diverse and emerging new species infecting multiple fruit crops.

To attract or to repel? Diversity, evolution and role of the “most peculiar organ” in the Cuscuta flower (dodder, Convolvulaceae)—the infrastaminal scales

Cuscuta (dodders) is the only genus in Convolvulaceae and the Solanales more broadly in which corolla-stamen structures called infrastaminal scales (IFS) have reached a high degree of elaboration and diversification in a great number of species. Historically, morphological diversity of IFS has provided some of the most useful taxonomic characters at the species-level. However, their function has not been determined. We have performed a comparative study of the IFS in 147 Cuscuta taxa using light, scanning and transmission electron microscopy, and results were analyzed in relation to a Cuscuta phylogeny obtained from a combined analysis of nuclear ITS and plastid trnL-F sequences. The morphology and histochemistry of scales and/or trichomes on the staminal filaments were also examined in several other Convolvulaceae genera to provide a preliminary foundation for homology interpretation. To test the hypothesis that the role of IFS in Cuscuta is connected to sexual reproduction, we analyzed the correlations between scale characters and previously published values for pollen/ovule ratios and the number of stomata found in the nectary at the base of the ovary. While the correlations at the level of the entire genus were very low, subgeneric partitions revealed a strong relationship between scale characters and sexual reproduction indicators in subgenus Monogynella. However, this connection declined drastically in the subgenera Cuscuta and Grammica. Our results strongly suggest that scales in Cuscuta evolved in connection to a modification of their function in the flower: from nectar protection and holding in the first diverged subgenus Monogynella, to ovary/ovule protection against herbivorous insects in the derived subgenera Cuscuta and Grammica. Thus at least in the case of the latter subgenera, the protective/repellent role of the IFS may conflict with the attractant/rewarding function of the nectary found at the base of the ovary. In subgenus Monogynella, IFS fimbriae are similar to uniseriate glandular hairs, with the secretory cells entirely exposed. In the subgenera Grammica and Cuscuta, the fimbriae become more complex, with an internal distal laticifer, and a precise mechanism of latex release. Our study elaborates further on the development and ultrastructure of scales in C. gronovii, and provides details on the anatomy of the IFS in other species. The new morphology and micromorphology data confirm the significance of these structures for species-level systematics.

An Endophytic Nodulisporium sp. from Central America Producing Volatile Organic Compounds with Both Biological and Fuel Potential

A Nodulisporium sp. (Hypoxylon sp.) has been isolated as an endophyte of Thelypteris angustifolia (Broadleaf Leaf Maiden Fern) in a rainforest region of Central America. It has been identified both on the basis of its morphological characteristics and by scanning electron microscopy as well as ITS sequence analysis. The endophyte produces volatile organic compounds (VOCs) that have both fuel (mycodiesel) and use for biological control of plant disease. When grown on potato dextrose agar, the organism uniquely produces a series of ketones, including acetone; 2-pentanone; 3-hexanone, 4-methyl; 3-hexanone, 2,4- dimethyl; 2-hexanone, 4-methyl, and 5-hepten, 2-one and these account for about 25% of the total VOCs. The most abundant identified VOC was 1,8 cineole, which is commonly detected in this group of organisms. Other prominent VOCs produced by this endophyte include 1-butanol, 2- methyl, and phenylethanol alcohol. Moreover, of interest was the presence of cyclohexane, propyl, which is a common ingredient of diesel fuel. Furthermore, the VOCs of this isolate of Nodulisporium sp. were selectively active against a number of plant pathogens, and upon a 24 h exposure caused death to Phytophthora palmivora, Rhizoctonia solani, and Sclerotinia sclerotiorum and 100% inhibition to Phytophthora cinnamomi with only slight to no inhibition of the other pathogens that were tested. From this work, it is becoming increasingly apparent that each isolate of this endophytic Nodulisporium spp., including the Daldina sp. and Hypoxylon spp. teleomorphs, seems to produce its own unique set of VOCs.

Pseudoidium javanicum, a new species of powdery mildew on Acalypha spp. from Indonesia

Pseudoidium javanicum is proposed as a new species based on analyses of 28S, ITS and IGS rDNA sequences, and morphological data. This new species was found on Acalypha wilkesiana var. marginata, A. argentea, and A. cristata collected from Cibodas Botanical Garden, Bogor (West Java Province, Indonesia). Our analyses showed that all these specimens have identical rDNA sequences and similar morphological characteristics. They form a distinct clade separated from other species of Erysiphaceae. Pseudoidium javanicum differs from Erysiphe acalyphae by having shorter conidiophores and foot cells 1–3 times as long as the 0–2 following cells. The conidial size of Ps. javanicum is also smaller than that of E. jatrophae.