S-ITS2 Region Research Articles

Morphological and molecular characterization, sexual reproduction, and pathogenicity of Setosphaeria rostrata isolates from rice leaf spot

Setosphaeria rostrata, a common plant pathogen causing leaf spot disease, affects a wide range of plant species, mainly grasses. Fungi were isolated from brown spots on rice leaves throughout Peninsular Malaysia, and 45 isolates were identified as Setosphaeria rostrata. The isolates were then characterized using morphological and molecular approaches. The mating type was determined using PCR amplification of the mating type alleles, and isolates of opposite mating types were crossed to examine sexual reproduction. Based on nuclear ribosomal DNA ITS1-5.8S-ITS2 region (ITS) and beta-tubulin (BT2) sequences, two phylogenetic trees were constructed using the maximum likelihood method; S. rostrata was clustered in one well-supported clade. Pathogenicity tests showed that S. rostrata isolates are pathogenic, suggesting that it is the cause of the symptoms. Mating-type analyses indicated that three isolates carried the MAT1-1 allele, and the other 42 isolates carried MAT1-2. After isolates with opposite mating types were crossed on Sach’s medium and incubated for 3 wk, six crosses produced pseudothecia that contained eight mature ascospores, and 12 other crosses produced numerous pseudothecia with no ascospores. To our knowledge, this is the first report on S. rostrata isolated from leaf spots on rice.

Multilocus phylogenetic reconstruction of the Clavariaceae (Agaricales) reveals polyphyly of agaricoid members

The genus Camarophyllopsis contains species with lamellate (agaricoid) basidiomes in the family Clavariaceae (Agaricales), a group otherwise dominated by club-like (clavarioid) or branched (coralloid) forms. Previous studies have suggested that species classified in Camarophyllopsis occur in two independent lineages. We reconstructed a multilocus phylogeny of the Clavaria-Camarophyllopsis-Clavicorona clade in the Clavariaceae using RNA polymerase II second largest subunit (rpb2), nuclear ribosomal 28S, and nuclear ribosomal ITS1-5.8S-ITS2 regions data and detected three independent groups of agaricoid fungi, including the genera Camarophyllopsis, Hodophilus, and Lamelloclavaria gen. nov, which distinctly differ in their pileipellis structure. In all, nine major lineages within the Clavaria-Camarophyllopsis-Clavicorona clade were recovered: Clavaria sensu stricto, Camarophyllopsis sensu stricto, Hodophilus, the Clavaria pullei clade, the Clavaria fumosa clade, Lamelloclavaria gen. nov., the Clavaria atrofusca clade, Holocoryne (= Clavaria sect. Holocoryne), and Clavicorona. Clavaria is paraphyletic and represented by five clades. Additional gene sampling is necessary to determine and confirm relatedness of these lineages before splitting Clavaria into additional genera.

Three rDNA Loci-Based Phylogenies of Tintinnid Ciliates (Ciliophora, Spirotrichea, Choreotrichida).

To improve understanding of diversity, phylogeny and evolution in tintinnid ciliates, it is essential to link multiple molecular markers with properly identified and documented morphospecies. Accordingly, 54 tintinnid morphospecies/isolates mainly from the Yellow and East China Seas were collected and analysed. Using single-cell approaches, sequences were obtained for three rDNA loci (18S, ITS1-5.8S-ITS2, D1-D5 region of 28S). Twenty-six tintinnid morphospecies (29 isolates) are documented by micrographs, measurements, morphologically described, and compared with the original species description. Three rDNA loci-based phylogenetic analyses were then performed for these identified isolates. Sequences from 25 unidentified species/isolates were also included in the comparison of the three rDNA loci. Ribosomal DNA genes of the genus Leprotintinnus were analysed for the first time, showing that Leprotintinnus was closely related to Tintinnopsis radix and branched distinctly apart from the family Tintinnidiidae. Four novel clades (VI to IX) of the Tintinnopsis complex emerged in the 18S genealogies. Analyses of the relative variability in the ITS and 28S regions vs. the 18S rDNA showed that the ITS1-5.8S-ITS2 and ITS2 regions well co-varied with the 18S rDNA when the variations of the latter were less than 3%, whereas at difference of less than 1%, no correlation was found between the compared loci. These findings highlight the difficulties in using variable locus-based cut-off divergences in circumscribing tintinnid morphospecies.

Occurrence of entomopathogenic fungi on insect pests of stored wheat and maize in Central and South Anatolia in Turkey

Naturally occurring fungal pathogens of stored-grain insect pests were identified and quantified using different insect sampling techniques in wheat and maize storage facilities in Central and South Anatolia in Turkey. Storage facilities were sampled by probing, trapping and visual inspection in five cities (Sanliurfa, Kahramanmaras, Adana, Mersin and Konya) from June to November 2013. Entomopathogenic fungi recovered from dead insects were identified to species level by sequencing the ITS1-5.8S-ITS2 region of the genomic DNA. Of the three species isolated, the majority were Beauveria bassiana (97 isolates), followed by Purpureocillium lilacinum (20 isolates). The third species, Beauveria varroae (9 isolates), is the first record on stored-product pest insects. Thirty-five isolates were from Tribolium spp., 29 from Sitophilus spp., 24 from Cryptolestes ferrugineus, 22 from Rhyzopertha dominica, 8 from Oryzaephilus surinamensis, 4 from Trogoderma granarium, 3 from Latheticus oryzae and 1 from a species of Cryptophagidae. The fungal infection of stored-grain pests did not vary significantly according to the time of sampling. A higher frequency of occurrence was recorded for Adana than the other cities and for Tribolium species than the other hosts. Grain samples taken by probing resulted in a higher frequency of fungal infection, but commodity type did not have a significant effect. The results demonstrated that (1) entomopathogenic fungi occurred at a low frequency, and (2) location, together with sampling technique, can affect their recovery. Further exploration of this ecosystem could yield important information for improving their use for management of stored-grain pests.

Three New Eremophilanes from a Ligularia Hybrid Collected in China

Three new eremophilanes were isolated from a sample of Ligularia collected in Yunnan province of China and their structures were determined. The chemical - constituents including eleven eremophilanes (major constituents: 6-acyloxyfuranoeremophilan-IOβ-ols) were similar to those of a hybrid of L. cyathiceps and L. subspicata, however, the base sequence of the ITS 1-5.8S-ITS2 region was typical of L. cyathiceps, indicating backcrossing.

First report of Aspergillus sp. causing postharvest fruit rot in cambuci (Campomanesia phaea) in Brazil

Cambuci (Campomanesia phaea), family Myrtaceae, is one of the most important endemic fruits of the Atlantic forest. In Brazil, the fruit is processed and consumed as jam, juice and ice cream. In October 2015, unusual fruit rot symptoms were observed five-ten days after harvest on cambuci kept at room temperature in packing houses in Salesópolis and Piracicaba, São Paulo State. Disease symptoms were soft lesions on the fruit surface with abundant dark grey mycelium and sporulation, and losses of up to 70% were observed. The pathogen was isolated on potato dextrose agar by sampling spores from the lesions. A representative monosporic isolate, CAM1USP was identified on the basis of morphological and molecular (rDNA-ITS) features. This isolate was stored in the mycological collection of the Laboratory of Epidemiology, University of São Paulo. When the isolate was incubated in the dark at 25°C for seven days, the colonies initially developed white mycelium without zonation and later were covered with a dense layer of dark brown or black radiate conidial heads (Fig. 1). Vesicles were subglobose to globose and measured 43.8 to 55.1 μm (n = 50). Conidia produced in vitro were brown to black, subglobose to globose and measured in diameter (4.7-) 5.6 (-6.4) μm (n = 50). All morphological features were similar to those of Aspergillus section Nigri (Varga et al., 2). To identify the species, DNA was extracted using a Promega Wizard genomic DNA purification kit (Madison, USA). Primer pairs ITS1 and ITS4 (White et al., 3) were used to amplify the ITS1-5.8S-ITS2 region. The resulting sequence of 523 bp (GenBank Accession No. KU751784) was compared to other sequences and was most similar to A. aculeatus (KP965728, 93%), A. japonicus (KP193133, 93%) and A. niger (KT726919, 92%). To test pathogenicity, ten cambuci fruits were disinfected with 0.5% sodium hypochlorite solution for one minute, wounded (3 mm deep) with a sterile histological needle and wound-inoculated with a 50 μl conidial suspension (106 conidia/ml). Ten control fruits were inoculated with sterile water. Fruits were maintained in a moisture chamber for 24 hours and kept at 25°C on the laboratory bench for one week. Two days after inoculation, all Aspergillus-inoculated fruits showed soft lesions that increased quickly in diameter, with abundant dark grey mycelium (Fig. 2). Aspergillus sp. was re-isolated from the lesions fulfilling Koch's postulates. Molecular and morphological characterisation and pathogenicity tests were repeated twice. The genus Aspergillus is known to cause postharvest diseases in fruit and seeds and toproduce mycotoxins in different crops (Palencia et al., 1). To our knowledge, this is the first report of Aspergillus sp. associated with postharvest fruit rot in cambuci and further studies will be necessary to fully resolve the taxonomy.

Analysis of the genetic diversity of Candida isolates obtained from diabetic patients and kidney transplant recipients.

Yeasts of the genus Candida have high genetic variability and are the most common opportunistic pathogenic fungi in humans. In this study, we evaluated the genetic diversity among 120 isolates of Candida spp. obtained from diabetic patients, kidney transplant recipients and patients without any immune deficiencies from Paraná state, Brazil. The analysis was performed using the ITS1-5.8S-ITS2 region and a partial sequence of 28S rDNA. In the phylogenetic analysis, we observed a consistent separation of the species C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. parapsilosis, C. metapsilosis and C. orthopsilosis, however with low intraspecific variability. In the analysis of the C. albicans species, two clades were formed. Clade A included the largest number of isolates (91.2%) and the majority of isolates from GenBank (71.4%). The phylogenetic analysis showed low intraspecific genetic diversity, and the genetic polymorphisms between C. albicans isolates were similar to genetic divergence found in other studies performed with isolates from Brazil. This low genetic diversity of isolates can be explained by the geographic proximity of the patients evaluated. It was observed that yeast colonisation was highest in renal transplant recipients and diabetic patients and that C. albicans was the species most frequently isolated.

Molecular phylogeny and species delimitation within the ciliate genus Spirostomum (Ciliophora, Postciliodesmatophora, Heterotrichea), using the internal transcribed spacer region

Morphological and molecular delimitation of Spirostomum species is currently under debate. We addressed species boundaries within the genus Spirostomum, using the ITS1-5.8S-ITS2 region and the secondary structure of the ITS2 molecule, and 18S and 28S (D1D2) sequences additionally. The Spirostomum ITS region is among the shortest within the ciliates hitherto studied. The Spirostomum ITS2 molecule matches the “ring model”, but exhibits only two helices radiating from a common loop. According to comparative analyses, they very likely correspond to helices II and III of other eukaryotes. Our phylogenetic analyses of the ITS region revealed a complex genealogical structure within the genus Spirostomum. However, boundaries among Spirostomum species could not be unambiguously determined either by phylogenetic trees, networks or sequence divergence cutoffs, because ITS2 sequences transcended species boundaries of the following morphospecies: S. ambiguum, S. minus, S. subtilis and S. teres. According to molecular diversity analysis, this is very likely caused by polymorphism in S. minus and S. teres, and by the lack of variability in S. ambiguum and S. subtilis. No compensatory base changes (CBCs) were detected in helices of the ITS2 molecule between different Spirostomum species, documenting that CBC analysis per se is not able to effectively discriminate Spirostomum species.

Isolation of the symbiotic fungus of Acromyrmex pubescens and phylogeny of Leucoagaricus gongylophorus from leaf-cutting ants

Leaf-cutting ants live in an obligate symbiosis with a Leucoagaricus species, a basidiomycete that serves as a food source to the larvae and queen. The aim of this work was to isolate, identify and complete the phylogenetic study of Leucoagaricus gongylophorus species of Acromyrmex pubescens. Macroscopic and microscopic features were used to identify the fungal symbiont of the ants. The ITS1-5.8S-ITS2 region was used as molecular marker for the molecular identification and to evaluate the phylogeny within the Leucoagaricus genus. One fungal symbiont associated with A. pubescens was isolated and identified as L. gongylophorus. The phylogeny of Leucoagaricus obtained using the ITS molecular marker revealed three well established monophyletic groups. It was possible to recognize one clade of Leucoagaricus associated with phylogenetically derived leaf-cutting ants (Acromyrmex and Atta). A second clade of free living forms of Leucoagaricus (non-cultivated), and a third clade of Leucoagaricus associated with phylogenetically basal genera of ants were also recognized. The clades corresponded to traditional taxonomic groups, and were differentiated by ecological habitats of different species.

The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA.

Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, andCandida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

Identification and characterization of Ditylenchus spp. populations from garlic in New York State, USA

Ditylenchus dipsaci is an important pathogen of onion and garlic and causes reductions in bulb quality. Between 2012 and 2014, 345 garlic bulb samples were tested for Ditylenchus spp. The average incidence of Ditylenchus spp. was 14.1 %. Twenty-five individuals from 31 Ditylenchus spp. populations were selected for identification by morphology, species-specific PCR, and sequencing of the ITS1-5.8S-ITS2 region. Thirty populations were identified by morphology and species-specific PCR as D. dipsaci. Sequencing of the ITS1-5.8S-ITS2 region of the ribosomal DNA found the populations were 99.7 to 100 % similar to each other, and 99.3 to 100 % similar to other D. dipsaci reference isolates from France, and California, USA. One population was identified as Ditylenchus sp. and was 97 % similar within the ITS1-5.8S-ITS2 region to D. destructor. This information will be useful for the monitoring of future pest outbreaks in garlic in New York State, USA, and the effectiveness of management recommendations.

DNA Barcoding Green Microalgae Isolated from Neotropical Inland Waters.

This study evaluated the feasibility of using the Ribulose Bisphosphate Carboxylase Large subunit gene (rbcL) and the Internal Transcribed Spacers 1 and 2 of the nuclear rDNA (nuITS1 and nuITS2) markers for identifying a very diverse, albeit poorly known group, of green microalgae from neotropical inland waters. Fifty-one freshwater green microalgae strains isolated from Brazil, the largest biodiversity reservoir in the neotropics, were submitted to DNA barcoding. Currently available universal primers for ITS1-5.8S-ITS2 region amplification were sufficient to successfully amplify and sequence 47 (92%) of the samples. On the other hand, new sets of primers had to be designed for rbcL, which allowed 96% of the samples to be sequenced. Thirty-five percent of the strains could be unambiguously identified to the species level based either on nuITS1 or nuITS2 sequences’ using barcode gap calculations. nuITS2 Compensatory Base Change (CBC) and ITS1-5.8S-ITS2 region phylogenetic analysis, together with morphological inspection, confirmed the identification accuracy. In contrast, only 6% of the strains could be assigned to the correct species based solely on rbcL sequences. In conclusion, the data presented here indicates that either nuITS1 or nuITS2 are useful markers for DNA barcoding of freshwater green microalgae, with advantage for nuITS2 due to the larger availability of analytical tools and reference barcodes deposited at databases for this marker.

Novel ophiostomatalean fungi from galleries of Cyrtogenius africus (Scolytinae) infesting dying Euphorbia ingens.

Euphorbia ingens trees have been dying in large numbers in the Limpopo Province of South Africa for approximately 15 years. The ambrosia beetle Cyrtogenius africus is often found infesting diseased and dying trees. The aim of this study was to identify the ophiostomatoid fungi occurring in the galleries of C. africus. Logs infested with this beetle were collected from the KwaZulu-Natal, Limpopo, Mpumalanga, and North West Provinces of South Africa. Fungi belonging to the Ophiostomatales were identified based on morphology and comparison of sequence data for the β-tubulin, ITS1-5.8S-ITS2 and LSU gene regions. A novel species of Ophiostoma and a novel genus in the Ophiostomatales were identified. Inoculation studies with these fungi produced lesions in the branches of healthy E. ingens trees.

First description of Candida nivariensis in Brazil: antifungal susceptibility profile and potential virulence attributes.

This study evaluated the antifungal susceptibility profile and the production of potential virulence attributes in a clinical strain of Candida nivariensis for the first time in Brazil, as identified by sequencing the internal transcribed spacer (ITS)1-5.8S-ITS2 region and D1/D2 domains of the 28S of the rDNA. For comparative purposes, tests were also performed with reference strains. All strains presented low planktonic minimal inhibitory concentrations (PMICs) to amphotericin B (AMB), caspofungin (CAS), and voriconazole. However, our strain showed elevated planktonic MICs to posaconazole (POS) and itraconazole, in addition to fluconazole resistance. Adherence to inert surfaces was conducted onto glass and polystyrene. The biofilm formation and antifungal susceptibility on biofilm-growing cells were evaluated by crystal violet staining and a XTT reduction assay. All fungal strains were able to bind both tested surfaces and form biofilm, with a binding preference to polystyrene (p < 0.001). AMB promoted significant reductions (≈50%) in biofilm production by our C. nivariensis strain using both methodologies. This reduction was also observed for CAS and POS, but only in the XTT assay. All strains were excellent protease producers and moderate phytase producers, but lipases were not detected. This study reinforces the pathogenic potential of C. nivariensis and its possible resistance profile to the azolic drugs generally used for candidiasis management.

Molecular Analysis of Terminalia spp. Distributed in Thailand and Authentication of Crude Drugs from Terminalia Plants.

Terminalia, a large genus of Combretaceae, is distributed in Tropical Asia, Africa, and America. Some Terminalia plants are used in folk medicine because they possess powerful medicinal properties. Dried fruits of Terminalia bellirica and Terminalia chebula are used as the main ingredient in Triphala, a famous polyherbal formulation in Ayurvedic medicine and Thai folk medicine, because of their laxative, detoxifying, and rejuvenating effects. To clarify the phylogenetic relationships of medicinal Terminalia species (T. bellirica, T. chebula, and T. catappa) and authenticate their crude drugs, "Samo" and Triphala, nucleotide sequencing alignments in the internal transcribed spacer one-two (ITS 1-2) regions of Terminalia plants collected in Thailand were performed. The amplified fragments of Terminalia species were approximately 800 bp in length. To compare these sequences and DDBJ registered data, a molecular phylogenetic tree was constructed. Phylogenetic analysis clearly separated the sequences into two groups: Asian Terminalia and African Terminalia with some exceptions. In the analyzed sequences, the length of the ITS1-5.8S-ITS2 region was 674 bp in T. chebula, and 677 bp in T. bellirica and T. catappa. Eighty-one single nucleotide polymorphisms (SNPs) and nine insertion-deletions (indels) were observed, and the nucleotide sequences of this region showed species-specific sequences. Based on these differences, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system (ARMS) were applied to identify medicinal Terminalia species. Moreover, the ARMS method was chosen for fingerprinting analysis of Samo crude drugs and Triphala formulations because it was a fast, cost-effective, and reproducible approach.

Genetic Variation among Fragmented Populations of &amp;lt;i&amp;gt;Atriplex halimus&amp;lt;/i&amp;gt; L. Using Start Codon Targeted (SCoT) and ITS1-5.8S-ITS2 Region Markers

Two forms of A. halimus shrubs: erect habit (A. halimus) and bushy habit shrub (A. schweinfurthii) are used naturally isolated by a considerable distance from each other and occupy the same area. To explore the effect of natural isolation on the genetic basis of the two forms, Start Codon Targeted (SCoT) and the phylogenetic relationships of A. halimus by sequencing ITS1-5.8S-ITS2 regions of the ribosomal DNA are used. Significant isolation-by-distance relationship was found (r = 0.62, P = 0.001). Soil factors did not influence molecular variations. The natural isolation of A. halimus habitats restricts gene flow among the populations and the observed high within-population genetic diversity (74.19%) in this species is best explained by its outcrossing behaviour, long-lived individuals and overlapping generations. The UPGMA analysis of the SCoT results showed that all the studied populations were divided into two discrete genetic groups with significant separation of the two forms in Burg El-Arab area (Populations 1 and 2) and insignificant separation between two forms in El-Hammam area (population 5 and 6). The sequencing of the ITS1-5.8S-ITS2 rDNA regions also showed the insignificant separation of the two A. halimus forms. We conclude that gene flow depending on habitat fragmentation was the main factor affecting the population genetic differentiation. We suggest that the two forms do not merit specific rank in presence of interference between the two forms and absence of a breeding barrier fail to separate the different populations when they become sympatric.

Fungi Causing Storage Rot of Apple Fruit in Integrated Pest Management System and their Sensitivity to Fungicides

Abstract Apple fruit rot can be caused by several fungi. In Northern Europe, the most common storage rot, Bull’s eye rot, is caused by Neofabraea spp., bitter rot by Colletotrichum spp., brown rot by Monilinia fructigena, grey mould is caused by Botrytis cinerea and Fusarium rot by several Fusarium species. Blue mold decay caused by Penicillium expansum is an important disease in several European countries. Incidence of different causal agents may vary depending on cultivar, climate during growing season and agricultural practices. The main objective of the study was to obtain baseline information about apple rot-causing fungi, their incidence during fruit storage and to evaluate the fungicide sensitivity of most of isolated fungal species. The study was performed during the storage period of apples after the growth season of 2013. Rotten apples were sorted in the storage and part of them was brought to the laboratory in order to obtain fungal isolates. Fungi were identified according to the morphological characteristics and sequencing of the ITS1-5.8S-ITS2 region. During storage in February and March the total percentage of rotten apples in various cultivars varied from 3.6 to 58.9%. All post-harvest diseases described in Northern Europe were detected. In part of the storehouses apple rot caused by Cadophora luteo-olivacea was observed. Alternaria spp. and Cladosporium spp. were detected on few apples as secondary infection agents. Using the most often isolated fungal species, sensitivity tests were performed against five commonly used fungicides. In general, the sensitivity of tested fungi to the fungicides was high with exception of several Neofabraea and Alternaria isolates.

ALTERNARIA ALTERNATA AS THE CAUSE OF BUD ROT ON GLOBE ARTICHOKE REPORTED FOR THE FIRST TIME IN GREECE

A new disease of globe artichoke (Cynara scolymus), affecting ca. 50% of the edible portion, was observed during 2011 in Peloponnese (southern Greece). On flower buds, symptoms started as small concentric dark-brown sunken spots, later coalescing and covering the entire surface of the buds, which rotted completely. Single-spore cultures on potato dextrose agar (PDA) gave rise to white colonies which turned to grayish-black and produced verrucose, echinulate or smooth conidia in long chains. Conidia had 1 to 6 transverse and 0 to 2 longitudinal septa and measured 10.4-26.0x7.8-15.6 μm (average 18.1x10.1 μm). These morphological characteristics conform to those of Alternaria alternata (Fr.) Keissl. (Simmons, 2007). The ITS1-5.8S-ITS2 region was amplified with primers ITS1 and ITS4 and sequenced (GenBank Accession No. KT071541). BLAST search revealed 100% homology with sequences of various A. alternata isolates (e.g., LN835252). Twenty healthy detached artichoke buds were sprayed with a spore suspension (106 spores/ml), covered with a polyethylene bag for 48 h and incubated in a growth chamber at 25oC. Ten days post inoculation, spots similar to those observed in the field developed on all inoculated buds, while control buds sprayed with sterile water remained symptomless. A. alternata was consistently reisolated from inoculated buds. Alternaria sp. has been reported on globe artichoke in Brazil, California, Mexico and South Africa (Farr and Rossman, 2015). This is the first report of A. alternata as the cause of bud rot on C. scolymus in Greece.

Sour rot of carrot caused by Geotrichum candidum in Japan

In September 2002, a severe sour rot was found on roots of postharvest carrots (Daucus carota L. var. sativus Hoffm) in Hokkaido Prefecture. The causal fungus was isolated from rotten carrots and identified as Geotrichum candidum Link. based on its morphology, growth characteristics, assimilation tests, banding pattern using species-specific primers, and sequences of the ITS1-5.8S-ITS2 region of the ribosomal DNA. The fungal isolates caused rot symptoms on inoculated carrot roots similar to those observed in the original samples. This is the first report of sour rot on carrot in Japan.

Expanded phylogenetic analyses of the class Heterotrichea (Ciliophora, Postciliodesmatophora) using five molecular markers and morphological data

Most studies of the molecular evolution of Heterotrichea have been based solely on the 18S-rDNA gene, which were inconsistent with morphological classification. Because of the limitations of single locus phylogenies and the recurring problem of lack of resolution of deeper nodes found in previous studies, we present hypotheses of the evolution of internal groups of the class Heterotrichea based on multi-loci analyses (18S-rDNA, 28S-rDNA, ITS1-5.8S-ITS2 region, COI and alpha-tubulin) and morphological data. Phylogenetic trees from protein coding gene data are presented for Heterotrichea for the first time. Phylogenetic analyses included Bayesian inference, maximum likelihood, maximum parsimony methods, and optimal trees were statistically compared to alternative topologies from the literature. Additionally, the Bayesian concordance approach (BCA algorithm) was used to assess the concordance factor between topologies obtained from isolated analyses. Because different loci may evolve at different rates, resulting in different gene topologies, we also estimated a species tree for Heterotrichea using the STAR coalescence-based method. The results show that: (1) single gene trees are inconsistent regarding the position of some heterotrichean families; (2) the concatenation of all data in a total-evidence tree improved the resolution of deep nodes among the heterotrichean families and genera; (3) the coalescent-based species tree is consistent with phylogenies based on the 18S-rDNA gene and shows Spirostomidae as the stem group of Heterotrichea; (4) however, the total-evidence tree suggests that the large Heterotrichea cluster is divided into nine lineages in which Peritromidae diverges at the base of the Heterotrichea tree.