Culturable Fungal Community of Pterocladiella capillacea in Keelung, Taiwan: Effects of Surface Sterilization Method and Isolation Medium

Fungi associated with macroalgae are less known when compared with those on wood in the marine environment. In this study, we assessed the diversity of fungi associated with the red alga Pterocladiella capillacea at Chao-Jin Park, Keelung, Taiwan. Algal segments of healthy and dead thalli were washed/sterilized with different solutions (sterile artificial seawater, 70% ethanol, and 4% sodium hypochlorite), plated on three different media (glucose-yeast extract-peptone seawater agar (GYPS), potato dextrose seawater agar (PDAS), and artificial seawater agar (SA)), and isolated as pure cultures. Identification was mainly based on BLAST search analysis of the internal transcribed spacers of rDNA (ITS). The highest isolation frequency (no. of segment with fungi/total no. of segment × 100) was in dead thalli (61.23%), thalli washed with seawater (88.38%), and thalli plated on GYPS (62.10%). A total of 3187 isolates were cultured, representing 129 taxa (in 67 genera); the higher species richness was isolated from healthy thalli (119 species), thalli washed with seawater (111 species), and on GYPS (112 species). Ascomycota (Eurotiales, Hypocreales, Capnodiales, Pleosporales, Xylariales) dominated the fungal community in P. capillacea with many basidiomycetous yeasts and few Mucoromycota. Aspergillus, Cladosporium, Penicillium (Ascomycota), and Rhodosporidium (Basidiomycota) were the dominant genera associated with the alga. The surface washing/sterilization schemes of algal thalli affected fungal diversity, but the isolation media used did not. While these genera are known producers of antimicrobial secondary metabolites, they might form a mutualistic relationship with P. capillacea by exchanging nutrients from photosynthesis for protection from microbial diseases.

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Eutypella parasitica and Other Frequently Isolated Fungi in Wood of Dead Branches of Young Sycamore Maple (Acer pseudoplatanus) in Slovenia

Forests ◽

10.3390/f11040467 ◽

2020 ◽

Vol 11 (4) ◽

pp. 467 ◽

Cited By ~ 4

Author(s):

Ana Brglez ◽

Barbara Piškur ◽

Nikica Ogris

Keyword(s):

Species Diversity ◽

Fungal Community ◽

Sampling Site ◽

Outer Part ◽

Similarity Index ◽

Asymptomatic Infection ◽

Fungal Species ◽

Acer Pseudoplatanus ◽

Pure Cultures ◽

Sycamore Maple

Eutypella parasitica R.W. Davidson and R.C. Lorenz is the causative agent of Eutypella canker of maple, a destructive disease of maples in Europe and North America. The fungus E. parasitica infects the trunk through a branch stub or bark wound. Because the fungal community may have an impact on infection and colonization by E. parasitica, the composition of fungi colonizing wood of dead branches of sycamore maple (Acer pseudoplatanus L.) was investigated in five sampling sites in Slovenia. Forty samples from each sampling site were collected between the November 2017 and March 2018 period. Isolations were made from the wood in the outer part of dead branches and from discoloured wood in the trunk that originated from a dead branch. Pure cultures were divided into morphotypes, and one representative culture per morphotype was selected for further molecular identification. From a total of 2700 cultured subsamples, 1744 fungal cultures were obtained, which were grouped into 212 morphotypes. The investigated samples were colonized by a broad spectrum of fungi. The most frequently isolated species were Eutypa maura (Fr.) Sacc., Eutypa sp. Tul. and C. Tul., Fusarium avenaceum (Fr.) Sacc., Neocucurbitaria acerina Wanas., Camporesi, E.B.G. Jones and K.D. Hyde and E. parasitica. In this study, we distinguished species diversity and the fungal community. There were no significant differences in the diversity of fungal species between the five sampling sites, and branch thickness did not prove to be a statistically significant factor in fungal species diversity. Nevertheless, relatively low Jaccard similarity index values suggested possible differences in the fungal communities from different sampling sites. This was confirmed by an analysis of similarities, which showed that the isolated fungal community distinctly differed between the five sampling sites and between the different isolation sources. Eutypella parasitica was isolated from all five investigated sampling sites, although Eutypella cankers were observed in only three sampling sites, indicating the possibility of asymptomatic infection.

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The genus Parasola in Pakistan with the description of two new species

MycoKeys ◽

10.3897/mycokeys.30.21430 ◽

2018 ◽

Vol 30 ◽

pp. 41-60 ◽

Cited By ~ 2

Author(s):

Shah Hussain ◽

Habib Ahma ◽

Sadiq Ullah ◽

Najam-Ul-Sehar Afshan ◽

Donald H. Pfister ◽

...

Keyword(s):

New Species ◽

Elongation Factor ◽

Molecular Data ◽

Internal Transcribed Spacers ◽

Side View ◽

Germ Pore ◽

5.8S Rdna ◽

Rdna Its ◽

Two New Species ◽

Morphological And Molecular Data

Parasola is a genus of small, veil-less coprinoid mushrooms in the family Psathyrellaceae (Agaricales). The genus is not well documented in Asia, specifically in Pakistan. In this study we describe two new species Parasolaglabra and P.pseudolactea from Pakistan, based on morphological and molecular data. Phylogeny based on three DNA regions: nuc rDNA region encompassing the internal transcribed spacers 1 and 2 along with the 5.8S rDNA (ITS), nuc 28S rDNA D1-D2 domains (28S) and translation elongation factor 1α gene (TEF1α) show that the new taxa are clustered in a clade formed by the members of section Parasola of genus Parasola. Parasolaglabra with grayish pileus, slightly depressed pileal disc, lamellae separated from the stipe by pseudocollarium, basidiospores 14.5–16.5 × 9.5–11.5 × 8.0–10.5 µm, in front view broadly ovoid to oblong, some with rhomboidal outline, in side view ellipsoid, with eccentric germ-pore of 1.5 µm diameter. Parasolapseudolactea with yellowish brown to dull brown pileus, disc indistinctly umbonate, lamellae free, pseudocollarium absent, basidiospores 13.5–14.5 × 10.5–12.0 × 9.5–10.5 µm, in face view rounded triangular to heart shaped, rarely ovoid to subglobose, in side view ellipsoid to oblong, with eccentric germ-pore of 1.5 µm diam. In addition to these new species, P.auricoma and P.lilatincta were also studied. Morphological descriptions for the new species and comparison with known Parasola species are provided. Our observations highlight the diversity of Parasola in northern Pakistan and further document the need for additional systematic focus on the region’s fungi.

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Effective medium for in vitro sprouting of the buds and multiplication of the plantlets, and identification of the fungal contaminant associated with the explants of Gnetum

GSC Biological and Pharmaceutical Sciences ◽

10.30574/gscbps.2021.16.2.0221 ◽

2021 ◽

Vol 16 (2) ◽

pp. 001-013

Author(s):

Abwe Mercy Ngone ◽

Lawrence Monah Ndam ◽

Rita Mungfu Njilar ◽

Doungous Oumar ◽

Thomas Eku Njock

Keyword(s):

Culture Medium ◽

Culture Media ◽

Fungal Growth ◽

Growth Media ◽

Fungal Contamination ◽

Surface Sterilization ◽

Pure Cultures ◽

Nodal Cuttings ◽

Baseline Information

Plant tissue culture requires the optimization of growth media. Gnetum, known locally in Cameroon as “Eru” is an indigenous gymnospermous vegetable with diverse medicinal, nutritional, cultural and socio-economic values. This resource is over-exploited and expected to neighboring countries, resulting to increased scarcity in the forest. Preliminary work on the in vitro culture of nodal cuttings was faced by the problem of fungal contamination. It was therefore necessary to isolate and identify the fungal contaminant, optimize the surface sterilization of field material and compose an appropriate medium for sprouting. Pure cultures of the fungus were obtained and grown on Potato Dextrose Agar (PDA) and Sabouraud Dextrose Agar (SDA). The identification was based on the appearance of the fungal growth on plates and also on the microscopic view. This was affected by the use of keys. Gnetum explants were disinfected with the various concentrations of disinfectants, preceded in some instances by pre-treatments, as well as incorporating fungicides in the culture medium. Two different culture media were employed: the Woody Plant Medium (WPM) and the Murashige and Skoog (MS) based establishment medium (Y-1). Gnetum was found to live in association with a complex of Microsporum species. The level of contamination of cultures was reduced from 100% to 40% when pre-treated before disinfection and even lower to 10% by incorporating fungicides in the medium. Sprouting was observed in WPM. This study provides baseline information on the in vitro propagation of Gnetum and thus opens up avenues for more research to be carried out in this field.

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First Report of Colletotrichum siamense Causing Anthracnose of Monstera deliciosa in Zhanjiang, China

Plant Disease ◽

10.1094/pdis-08-20-1839-pdn ◽

2020 ◽

Author(s):

Yue Lian Liu ◽

Jian Rong Tang ◽

Yu Han Zhou

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Pathogenicity Test ◽

Sterile Water ◽

Single Spore ◽

First Report ◽

Rdna Its ◽

Colletotrichum Siamense ◽

Monstera Deliciosa ◽

Pure Cultures

Monstera deliciosa Liebm is an ornamental foliage plant (Zhen et al. 2020De Lojo and De Benedetto 2014). In July of 2019, anthracnose lesions were observed on leaves of M. deliciosa cv. Duokong with 20% disease incidence of 100 plants at Guangdong Ocean University campus (21.17N,110.18E), Guangdong Province, China. Initially affected leaves showed chlorotic spots, which coalesced into larger irregular or circular lesions. The centers of spots were gray with a brown border surrounded by a yellow halo (Supplementary figure 1). Twenty diseased leaves were collected for pathogen isolation. Margins of diseased tissue was cut into 2 × 2 mm pieces, surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite (NaOCl) for 60 s, rinsed three times with sterile water before isolation. Potato dextrose agar (PDA) was used to culture pathogens at 28℃ in dark. Successively, pure cultures were obtained by transferring hyphal tips to new PDA plates. Fourteen isolates were obtained from 20 leaves. Three single-spore isolates (PSC-1, PSC-2, and PSC-3) were obtained ,obtained, which were identical in morphology and molecular analysis (ITS). Therefore, the representative isolate PSC-1 was used for further study. The culture of isolate PSC-1 on PDA was initially white and later became cottony, light gray in 4 days, at 28 °C. Conidia were single celled, hyaline, cylindrical, clavate, and measured 13.2 to 18.3 µm × 3.3 to 6.5 µm (n = 30). Appressoria were elliptical or subglobose, dark brown, and ranged from 6.3 to 9.5 µm × 5.7 to 6.5 µm (n = 30). Morphological characteristics of isolate PSC-1 were consistent with the description of Colletotrichum siamense (Prihastuti et al. 2009; Sharma et al. 2013). DNA of the isolate PSC-1 was extracted for PCR sequencing using primers for the rDNA ITS (ITS1/ITS4), GAPDH (GDF1/GDR1), ACT (ACT-512F/ACT-783R), CAL (CL1C/CL2C), and TUB2 (βT2a/βT2b) (Weir et al. 2012). Analysis of the ITS (accession no. MN243535), GAPDH (MN243538), ACT (MN512640), CAL (MT163731), and TUB2 (MN512643) sequences revealed a 97-100% identity with the corresponding ITS (JX010161), GAPDH (JX010002), ACT (FJ907423), CAL (JX009714) and TUB2 (KP703502) sequences of C. siamense in GenBank. A phylogenetic tree was generated based on the concatenated sequences of ITS, GAPDH, ACT, CAL, and TUB2 which clustered the isolate PSC-1 with C. siamense the type strain ICMP 18578 (Supplementary figure 2). Based on morphological characteristics and phylogenetic analysis, the isolate PSC-1 associated with anthracnose of M. deliciosa was identified as C. siamense. Pathogenicity test was performed in a greenhouse at 24 to 30oC with 80% relative humidity. Ten healthy plants of cv. Duokong (3-month-old) were grown in pots with one plant in each pot. Five plants were inoculated by spraying a spore suspension (105 spores ml-1) of the isolate PSC-1 onto leaves until runoff, and five plants were sprayed with sterile water as controls. The test was conducted three times. Anthracnose lesions as earlier were observed on the leaves after two weeks, whereas control plants remained symptomless. The pathogen re-isolated from all inoculated leaves was identical to the isolate PSC-1 by morphology and ITS analysis, but not from control plants. C. gloeosporioides has been reported to cause anthracnose of M. deliciosa (Katakam, et al. 2017). To the best of our knowledge, this is the first report of C. siamense causing anthracnose on M. deliciosa in ChinaC. siamense causes anthracnose on a variety of plant hosts, but not including M. deliciosa (Yanan, et al. 2019). To the best of our knowledge, this is the first report of C. siamense causing anthracnose on M. deliciosa, which provides a basis for focusing on the management of the disease in future.

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Phylogenetic Analysis and Molecular Diversity of Capsicum Based on rDNA-ITS Region

Horticulturae ◽

10.3390/horticulturae6040087 ◽

2020 ◽

Vol 6 (4) ◽

pp. 87

Author(s):

Kumpei Shiragaki ◽

Shuji Yokoi ◽

Takahiro Tezuka

Keyword(s):

Genetic Diversity ◽

Phylogenetic Analysis ◽

Wild Species ◽

Morphological Characteristics ◽

Its Region ◽

Internal Transcribed Spacers ◽

Nuclear Ribosomal Dna ◽

Rdna Its ◽

Rdna Its Region ◽

Genus Capsicum

The genus Capsicum is comprised of 5 domesticated and more than 30 wild species. The region of nuclear ribosomal DNA internal transcribed spacers (rDNA-ITS) has widely been used for species identification, but has rarely been used in Capsicum. In this study, the evaluation of genetic diversity and a phylogenetic analysis were conducted using rDNA-ITS of 28 Capsicum accessions, including five domesticated and two wild species. We surveyed six conventional keys of domesticated species and another five traits in Capsicum accessions. Specific morphological characteristics were found in C. annuum, C. baccatum, and C.pubescens. Three subclones of each accession were sequenced, and rDNA-ITS polymorphisms were detected in all accessions excluding C. annuum, suggesting that incomplete concerted evolution occurred in rDNA-ITS of Capsicum. The genetic diversity was evaluated using nucleotide polymorphism and diversity. C. annuum had the lowest genetic diversity of all species in this study. The phylogenetic tree formed a species-specific clade for C. annuum, C. baccatum, and C. pubescens. The C. chinense clade existed in the C. frutescens clade, implying that it was a cultivated variant of C. frutescens. C. chacoense likely belonged to the C. baccatum complex according to its morphologic and genetic features. This study indicated that the rDNA-ITS region can be used for simple identification of domesticated Capsicum species.

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Molecular identification and artificial cultivation of a wild isolate of oyster mushroom in Albania

Italian Journal of Agronomy ◽

10.4081/ija.2016.704 ◽

2016 ◽

Vol 10 (1s) ◽

pp. 35

Author(s):

Jordan Merkuri ◽

Stefania Mirela Mang ◽

Ippolito Camele ◽

Magdalena Cara ◽

Gian Luigi Rana

Keyword(s):

Its Region ◽

Edible Mushroom ◽

Biological Efficiency ◽

Potato Dextrose Agar Medium ◽

Wild Isolate ◽

Rdna Its ◽

Pure Cultures ◽

Rdna Its Region ◽

And Diffusion ◽

Artificial Cultivation

Basidiomata of a wild mushroom macroscopically recognised as Pleurotus ostreatus were observed on an oak trunk in a mixed wood of northern Albania. Pure cultures of the fungus were then obtained on potato-dextrose-agar medium. Molecular analyses of genomic DNA of the fungus confirmed its identification. The rDNA ITS region nucleotide sequence of the studied Pleurotacea matched at 99% those of two P. ostreatus strains already present in NCBI GenBank database. The rDNA ITS nucelotide sequences of two pure cultures of the Albanian P. ostreatus were deposited in EMBL database under the accession numbers LN849458 and LN849459. One of the fungus isolates was subsequently cultivated under protected and semi-natural conditions. Productivity and biological efficiency of the Albanian P. ostreatus ranged from about 10% to 16% and from 33 to 53.33%, respectively. This seems to be the first report on the artificial cultivation of P. ostreatus in Albania and could have, in the next future, a high economic impact on development and diffusion of this important edible mushroom over the country.

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How to resolve cryptic species of polypores: an example in Fomes

IMA Fungus ◽

10.1186/s43008-019-0016-4 ◽

2019 ◽

Vol 10 (1) ◽

Author(s):

Ursula Peintner ◽

Regina Kuhnert-Finkernagel ◽

Viana Wille ◽

Franco Biasioli ◽

Anton Shiryaev ◽

...

Keyword(s):

Cryptic Species ◽

Growth Rates ◽

Species Delimitation ◽

Its Region ◽

Morphological Characters ◽

Rdna Its ◽

Pure Cultures ◽

Rdna Its Region ◽

The Mediterranean ◽

Physiological Characters

Abstract Species that cannot be easily distinguished based on morphology, but which form distinct phylogenetic lineages based on molecular markers, are often referred to as cryptic species. They have been proposed in a number of fungal genera, including the basidiomycete genus Fomes. The main aim of this work was to test new methods for species delimitation in cryptic lineages of polypores, and to define useful characters for species identification. A detailed examination of a number of different Fomes strains that had been collected and isolated from different habitats in Italy and Austria confirmed the presence of distinct lineages in the Fomes fomentarius clade. Our zero hypothesis was that the Mediterranean strains growing on Quercus represent a species which can be delimited based on morphological and physiological characters when they are evaluated in statistically relevant numbers. This hypothesis was tested based on phylogenetic analysis of the rDNA ITS region, morphological characters of basidiomes and pure cultures, growth rates and optimum growth temperature experiments, mycelial confrontation tests, enzyme activity tests and volatile organic compound (VOC) production. The Mediterranean lineage can unambiguously be delimited from F. fomentarius. A syntype of an obscure and previously synonymized name, Polyporus inzengae, represents the Mediterranean lineage that we recognize as Fomes inzengae, a distinct species. The rDNA ITS region is useful for delimitation of Fomes species. Moreover, also a variety of morphological characters including hymenophore pore size, basidiospore size, and diameter of skeletal hyphae are useful delimiting characters. The ecology is also very important, because the plant host appears to be a central factor driving speciation. Physiological characters turned also out to be species-specific, e.g. daily mycelial growth rates or the temperature range of pure cultures. The production of VOCs can be considered as a very promising tool for fast and reliable species delimitation in the future.

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Phylogenetic relationships in the Mediterranean Helichrysum (Asteraceae, Gnaphalieae) based on nuclear rDNA ITS sequence data

Australian Systematic Botany ◽

10.1071/sb03031 ◽

2004 ◽

Vol 17 (3) ◽

pp. 241 ◽

Cited By ~ 33

Author(s):

M. Galbany-Casals ◽

N. Garcia-Jacas ◽

A. Susanna ◽

L. Sáez ◽

C. Benedí

Keyword(s):

Sequence Data ◽

Cladistic Analysis ◽

Mediterranean Area ◽

Internal Transcribed Spacers ◽

Nuclear Rdna ◽

Generic Delimitation ◽

Rdna Its ◽

The Mediterranean ◽

Sectional Classification

The internal transcribed spacers ITS1 and ITS2 of the nuclear rDNA were sequenced for 41 Helichrysum species (Gnaphalieae), focusing on the Mediterranean group of species, together with eight representatives of other genera of the Gnaphalieae, in order to check the hypothesised monophyly of the Mediterranean Helichrysum group and the correspondence of the sequence data with its traditional sectional classification. The cladistic analysis of sequence data supports monophyly of the Mediterranean Helichrysum excluding H. frigidum and H. montelinasanum. The traditional classification of the Mediterranean species into two sections, Helichrysum and Virginea, is not supported, whereas a group constituted by species from the west Mediterranean area is shown as a moderately supported monophyletic clade in the strict consensus tree. Other results also show and confirm the complexity, still not satisfactorily resolved, of the Helichrysum generic delimitation: Pseudognaphalium luteoalbum appears merged in Helichrysum whereas Helichrysum dasyanthum appears more related to Anaxeton laeve than to any Helichrysum species.

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A study of variation in rDNA ITS regions shows that two haplotypes coexist within a single aphid genome

Genome ◽

10.1139/g98-030 ◽

1998 ◽

Vol 41 (3) ◽

pp. 337-345 ◽

Cited By ~ 35

Author(s):

Brian Fenton ◽

Gaynor Malloch ◽

Florence Germa

Keyword(s):

Green Peach Aphid ◽

Aphid Species ◽

Internal Transcribed Spacers ◽

Rdna Its ◽

Large Deletions ◽

Hot Start ◽

Pcr Products ◽

Pcr Conditions

We report variation in the rDNA internal transcribed spacers (ITSs) of aphid species, the first for these insects. Variation at 6 sites within ITS1 sequences of the green peach aphid, Myzus persicae, identified two haplotypes coexisting within the same individuals, indicating that molecular drive has not homogenised different copies of rDNA. During this study, we found that PCR can cause a precise 58-bp loss in the amplified copies of an ITS haplotype (type 1). This occurs in all detectable copies under routine PCR conditions, at different annealing temperatures and with Pfu and Taq polymerases. In addition, "hot-start" PCR exclusively copied a different, rare haplotype (type 2). These observations have important considerations for using PCR, as large deletions in PCR products may not reflect real deletions in the genome, and changes in PCR conditions may be needed to copy cryptic haplotypes.Key words: PCR, aphid, ITS, variation, selection.

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Diversity of mangrove fungal endophytes from selected mangrove species of coastal Kenya

Western Indian Ocean Journal of Marine Science ◽

10.4314/wiojms.v20i1.11 ◽

2021 ◽

Vol 20 (1) ◽

pp. 125-136

Author(s):

Helen M. Kiti ◽

Cosmas N. Munga ◽

Josiah O. Odalo ◽

Paul M. Guyo ◽

Cromwell M. Kibiti

Keyword(s):

Fungal Community ◽

Fungal Endophytes ◽

Avicennia Marina ◽

Community Diversity ◽

Colonization Rate ◽

Bioactive Metabolites ◽

Mangrove Species ◽

Red Mangrove ◽

Pure Cultures ◽

Gazi Bay

Endophytes are bacteria or fungi living asymptomatically in the internal tissues of plants. They are symbiotic in nature and can be exploited for novel bioactive metabolites with applications in agriculture, medicine and industry. Mangrove fungal endophytes from the marine environment are abundant and have been recognized as sources of bioactive natural products. The study was designed to isolate, purify and identify mangrove fungal endophytes from selected common mangrove species of Gazi Bay, Tudor and Mida creek on the Kenya coast. The colonization rate and isolation rate of the mangrove fungal endophytes were determined. The studied mangrove species were Rhizophora mucronata (red mangrove), Sonneratia alba (mangrove apple), Avicennia marina (grey or white mangrove), and Ceriops tagal (spurred mangrove). Samples from twigs of these mangrove species were collected and analyzed using standard methods. Isolation of pure cultures of the endophytes was performed using Potato Dextrose Agar (PDA) incubated at 28 ± 1ºC for 5 days. The fungal isolates were identified under a light microscope based on colony morphology characteristics, type and presentation of conidiophores and conidia. A total of 18 different mangrove fungal endophytes were identified and these belonged to 5 genera. These were Aspergillus, Penicillium, Fusarium, Cephalosporium and Blastomyces, with Aspergillus being the most dominant genus. Tudor Creek recorded the highest fungal community diversity (H’ = 1.35) and Gazi Bay had the lowest diversity (H’ = 0.45). Fungal community similarity based on the identified genera was highest between Gazi Bay and Mida Creek (0.80) and lowest between Tudor Creek and Mida Creek (0.57). The selected mangrove species recorded a colonization rate of endophytic fungi of between 38.9 – 94.4 % with the highest habitation being associated with S. alba and C. tagal. There were differences and similarities in the colonization rate within mangrove species across study sites. Findings of this study have confirmed that the selected mangrove species exhibit high diversity of fungal endophytes with host recurrence and spatial heterogeneity.

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