Β-tubulin Sequences Research Articles

A new species of Acrophialophora from Guizhou Province, China

Acrophialophora liboensis, a new fungus from soil samples in Libo County, Guizhou Province, China, is illustrated and described on the basis of morphological and molecular sequences data. Phylogenetic analysis based on internal transcribed spacer (ITS) and β-tubulin sequences demonstrated that A. liboensis is a distinct species closely related to A. cinerea and A. furcata. Morphologically, A. liboensis is characterized by solitary and lateral phialides tapering into thin necks and long chains of ellipsoidal or oval conidia. The ex-type living culture has been deposited in CGMCC, Beijing City, China.

Diatrypella tectonae and Peroneutypa mackenziei spp. nov. (Diatrypaceae) from northern Thailand

Two novel species of Diatrypaceae, Diatrypella tectonae and Peroneutypa mackenziei, were collected from dead wood in northern Thailand. The new species are introduced in this paper, with evidence from morphology and phylogenetic analyses. The new taxa are described, illustrated and compared with related taxa. Phylogenetic analyses of combined ITS and partial β-tubulin (β-tub) sequence data support their natural placements in the genera Diatrypella and Peroneutypa as new species.

FIRST REPORT OF DIPLODIA SERIATA AS CAUSAL AGENT OF ALMOND TREE BRANCH DIEBACK IN TUNISIA

A branch dieback of almond trees (cv. Achaak) was noticed in 2016 in the region of Sfax. 10% of trees showed dead branches with sunken areas. Infected tissues were abnormally dark and cross sections revealed a light-brown and irregularly shaped canker developing from the graft-union. Numerous pycnidia were observed on the surface of infected branches with severe gummosis on the buds. To identify the pathogen, 35 small pieces of infected tissues were plated on potato dextrose agar. After incubation at 25°C for 5 days, 15 isolates were consistently isolated. Colonies were grayish white, producing dark melanised hyphae and pycnidia after 10 days of incubation. Conidia were first hyaline, and then turned brown as they matured. They were aseptate, rounded at both ends with thick melanised cell walls, 19 to 25.7 × 9.5 to 11.03 µm. Based on colony characteristics, all isolates were identified as Diplodia seriata (Phillips et al., 2007). Molecular identification was performed by sequencing the ITS, the EF-1-α and the β-tubulin genes. BLAST searches of ITS (KY275259), EF-1-α (KY284865) and β-tubulin (KY275260) sequences, respectively revealed 98%, 99% and 100% identity to Diplodia seriata. Pathogenicity tests were performed on detached stems, wounded with a scalpel and inoculated with mycelial plugs (Yan et al., 2011). Controls were inoculated with sterile agar plugs. Inoculated stems were placed in polyethylene boxes and incubated at 25°C for 30 days. While mock-infected stems remained healthy, inoculated stems developed brown discoloration, with small dark pycnidia growing on the surface. The pathogen was isolated from inoculated stems, thus fulfilling Koch's postulates. To our knowledge, this is the first report of canker disease caused by Diplodia seriata in Tunisia

Pyrenochaetopsis tabarestanensis (Cucurbitariaceae, Pleosporales), a new species isolated from rice farms in north Iran

A pyrenochaeta-like species was isolated from soil in rice farms in Mazandaran Province (formerly Tabarestan), Iran. Phylogenetic analyses of SSU, LSU, ITS, β-tubulin and actin sequence data showed that the new collection clustered with species in Pyrenochaetopsis sensu stricto. In morphology, phylogeny and physiological characters, the new strains are distinct from other species, thus, they are introduced as a new species viz. P. tabarestanensis. Illustrations, descriptions and taxonomic notes are provided for P. tabarestanensis. Additionally, the phylogenetic relationships of Pyrenochaeta and Pyrenochaetopsis species in Cucurbitariaceae is discussed.

First Report of Colletotrichum fructicola Causing Anthracnose on Annona Leaves in Brazil

HomePlant DiseaseVol. 101, No. 2First Report of Colletotrichum fructicola Causing Anthracnose on Annona Leaves in Brazil PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Colletotrichum fructicola Causing Anthracnose on Annona Leaves in BrazilJ. F. O. Costa, R. Ramos-Sobrinho, T. P. Chaves, J. R. A. Silva, D. B. Pinho, I. P. Assunção, and G. S. A. LimaJ. F. O. CostaSearch for more papers by this author, R. Ramos-SobrinhoSearch for more papers by this author, T. P. ChavesSearch for more papers by this author, J. R. A. SilvaSearch for more papers by this author, D. B. PinhoSearch for more papers by this author, I. P. AssunçãoSearch for more papers by this author, and G. S. A. LimaSearch for more papers by this authorAffiliationsAuthors and Affiliations J. F. O. Costa R. Ramos-Sobrinho T. P. Chaves J. R. A. Silva , Setor de Fitossanidade/Centro de Ciências Agrárias, Universidade Federal de Alagoas, Rio Largo, AL, Brazil, 57100-000 D. B. Pinho , Departamento de Fitopatologia, Universidade de Brasília, Brazil, 70910-900 I. P. Assunção G. S. A. Lima , Setor de Fitossanidade/Centro de Ciências Agrárias, Universidade Federal de Alagoas, Rio Largo, AL, Brazil, 57100-000. Published Online:21 Nov 2016https://doi.org/10.1094/PDIS-06-16-0927-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Anthracnose, caused by different Colletotrichum species, is major disease on Annona spp. (Kamei et al. 2014). This disease has been mainly associated with the Colletotrichum gloeosporioides complex (Kamei et al. 2014). In June 2012, two typical isolates of the C. gloeosporioides complex were obtained from symptomatic leaves (dark, circular, and necrotic lesions) of plants located in the counties of Maragogi and Estrela de Alagoas, Alagoas state, Brazil. Single-conidium cultures were obtained on potato dextrose agar (PDA) for both isolates, and deposited at the Coleção de Culturas de Fungos Fitopatogênicos da Universidade Federal de Alagoas (codes COUFAL1111 and COUFAL7777). Colonies on PDA were light gray with whitish edges, and cottony. The underside of the colony was uniformly gray presenting sectors toward the edges with a growth rate of 9.0 mm/day. Conidia were cylindrical, aseptate, hyaline, rounded at both ends, and 10.5 to 22.5 µm long and 3 to 6.5 µm wide. The morphological characteristics were consistent with descriptions of C. gloeosporioides sensu lato (Weir et al. 2012). To confirm the identity, partial sequences of manganese-superoxide dismutase (SOD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS), calmodulin (CAL), β-tubulin 2 (TUB2), actin (ACT), and internal transcribed spacer (ITS) (accession nos. KX013517 to KX013530) were used in a search in the Q-bank fungi database and retrieved C. fructicola with 99 to 100% identities. In addition, Bayesian inference analysis using concatenated sequences (TUB2, ACT, CAL, CHS, GAPDH, ITS, and SOD) placed the Colletotrichum isolates reported here in a same clade with other C. fructicola specimens (study S19095 deposited in TreeBASE). To confirm pathogenicity, a conidial suspension (106 conidia/ml) in water was prepared from a 15-day-old culture (grown at 25°C) and sprayed on asymptomatic leaves of soursop (A. muricata L.) and sugar apple (A. squamosa L.). On control leaves, only sterilized water was used. Leaves were kept for 2 days in a humid chamber at 25°C. After 5 days, lesions were observed on 100% of the inoculated leaves. To fulfill Koch’s postulates, the fungus C. fructicola was successfully reisolated from inoculated leaves. No disease symptoms were observed on the control leaves. Other Colletotrichum species were recorded on Annona spp. (Kamei et al. 2014), but no isolate was genetically similar to C. fructicola. This pathogen was previously reported only on apple, cassava, and mango in Brazil (Bragança et al. 2016; Lima et al. 2013; Velho et al. 2015). To our knowledge, this is the first report of C. fructicola causing anthracnose on leaves of Annona spp. in Brazil.

Multilocus molecular identification and phylogenetic analysis of Colletotrichum tamarilloi as the causal agent of Tamarillo (Solanum betaceum) anthracnose in the Ecuadorian highlands

Anthracnose caused by Colletotrichum spp., is one of the most serious diseases affecting tamarillo (Solanum betaceum) production in the Ecuadorian highlands. The objective of this study was to characterise Colletotrichum isolates obtained from tamarillo to clarify its taxonomic and phylogenetic position. Based on phenotypic and morphologic characterisation, the isolates of this study were consistently grouped within the Colletotrichum acutatum complex. Multilocus molecular phylogenetic analysis based on combined sequences of actin (ACT), β-tubulin (TUB2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sequences using Bayesian probabilities, indicated that 16 of 20 isolates from Imbabura, Cotopaxi, Tungurahua, Bolivar, Chimborazo, Azuay and Loja provinces, belonged to C. tamarilloi. This study represents the first reported case of anthracnose of S. betaceum caused by C. tamarilloi in the Ecuadorian highlands. Interestingly, pathogenicity tests and multilocus molecular analysis revealed a new infraspecific and more aggressive species formed by four isolates obtained from the Pichincha province. These findings probably provided interesting information related to mutant isolates and a possible description of a new pathogenic species affecting tamarillo fruits.

De novo STAT3 Mutation in a Patient with Fatal, Treatment-Refractory Sino-orbital Aspergillosis

Background Signal transducer and activator of transcription 3 (STAT3) loss-of-function mutations in humans result in Job’s syndrome, characterized by elevated IgE, bacterial infections, chronic mucocutaneous candidiasis, pulmonary aspergillosis due to structural lung disease, and connective tissue abnormalities.Methods Whole exome sequencing (WES) was performed on a putatively immunocompetent patient with sino-orbital aspergillosis and his parents. Evaluation of STAT3 phosphorylation (pSTAT3) was performed by flow cytometry on peripheral blood mononuclear cells after IL-6 stimulation.ResultsA 37-year-old-male with a history of eosinophilic esophagitis developed a locally invasive sino-orbital infection with progressive cavernous sinus involvement associated with facial numbness, diplopia, and visual loss. Biopsy showed chronic inflammation and invasive fungal hyphae. Cultures grew an isolate of Aspergillus fumisynnematus that was identified by morphology and the sequences of β-tubulin and Mcm7. MICs (in µg/ml) for the isolate were: micafungin <=0.015, amphotericin 2, posaconazole 0.25, terbinafine 0.06, and voriconazole 0.25. The patient underwent multiple surgical debridements and was treated over time with various antifungals (amphotericin B, micafungin, terbinafine, voriconazole, posaconazole), adjunct cytokines (IFN-γ, GM-CSF), and hyperbaric oxygen. However, the infection progressed into the right middle cranial fossa and meninges and the patient died 1 year after symptoms began. WES revealed a de novo splice-site mutation in STAT3 (c.1140-3C>G). cDNA sequencing showed nonsense mediated decay of the affected allele. No mutations in CARD9 or NADPH oxidase subunits were found; a DHR test was normal. The patient had normal blood myeloid cell subsets. Serum IgE level was elevated at 833 IU/ml. After stimulation with IL-6, the patient’s memory CD4+ T cells and CD11c+ myeloid cells had reduced pSTAT3 levels compared with control cells. Cellular analysis of SOCS3, a STAT3-dependent downstream target, is underway to evaluate for functional STAT3 haploinsufficiency.Conclusion A novel de novo STAT3 splice site mutation results in impaired pSTAT3, and is associated with elevated IgE, eosinophilic esophagitis, and sino-orbital aspergillosis without other common features of Job’s syndromeDisclosures All authors: No reported disclosures.

Morphological and Molecular Diversity of Aspergillus From Corn Grain Used as Livestock Feed

The study of Aspergillus from corn grains used as livestock feed is important to ensure the safety of the grains as the occurrence of Aspergillus in the corn grain can give an indication of mycotoxin being produced. Morphological and molecular identifications were applied to identify Aspergillus isolated from corn grains used as livestock feed. Morphologically, six species were tentatively identified, namely Aspergillus niger (Groups I and II), Aspergillus flavus, Aspergillus oryzae, Aspergillus fumigatus, Aspergillus clavatus and Aspergillus terreus. Isolates of A. niger was divided into two groups based on slight differences of their colony appearances. Molecular identification using internal transcribed spacer and β-tubulin sequences supported morphological identification except isolates for A. niger Group II isolates, which were molecularly identified as Aspergillus tubingensis. Neighbour-joining phylogenetic tree showed that isolates from the same species were grouped in the same clade. The present study showed that diverse species of Aspergillus are prevalent in corn grain used as livestock feed. It is also necessary to correctly identify the Aspergillus species to employ correct treatment of contaminated corn grains. The occurrence of well-known toxigenic species such as A. niger, A. flavus and A. fumigatus suggested the possible risk of mycotoxin contamination of the corn grain.

Anthostomelloides krabiensis gen. et sp. nov. (Xylariaceae) from Pandanus odorifer (Pandanaceae)

An Anthostomella-like taxon was obtained from Pandanus odorifer (Forssk.) Kuntze (Pandanaceae) collected in Krabi Province in Thailand. Morphological data plus phylogenetic analyses of combined ITS, LSU, RPB2, and β-tubulin sequence data clearly separate this Anthostomella-like taxon from other known genera in Xylariaceae. In this paper, we introduce this taxon as a new genus, Anthostomelloides, in the family Xylariaceae, with A. krabiensis as the type. A detailed morphological description, phylogenetic tree, photomicrographs of A. krabiensis, keys to Anthostomella-like genera, and a comparison of A. krabiensis with the morphologically similar taxa in Xylariaceae are provided.

First Report of Nothophoma quercina Causing Shoot Canker on Chaenomeles sinensis in Korea

HomePlant DiseaseVol. 100, No. 12First Report of Nothophoma quercina Causing Shoot Canker on Chaenomeles sinensis in Korea PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Nothophoma quercina Causing Shoot Canker on Chaenomeles sinensis in KoreaY. H. Yun and M. H. OhY. H. YunSearch for more papers by this author and M. H. OhSearch for more papers by this authorAffiliationsAuthors and Affiliations Y. H. Yun , Department of Microbiology, Dankook University, Cheonan 31116, Republic of Korea M. H. Oh , Department of NanoBioMedical Science, Dankook University, Cheonan 31116, Republic of Korea. Published Online:4 Oct 2016https://doi.org/10.1094/PDIS-06-16-0870-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Chaenomeles sinensis Koehne, Rosaceae family, is widely distributed in Korea, China, and Japan. It has been used as a folk remedy in the treatment of asthma, the common cold, and dry throat. Recently, the medicinal properties of fruit of C. sinensis have been validated by numerous pharmacological studies on anti-influenza, antioxidant, and antidiabetic (Chun et al. 2012). During summer 2015, shoot canker was observed on C. sinensis in Cheonan (36°50′15.0″ N, 127°10′01.8″ E), Korea. Necrotic disease incidence was nearly 5% on young shoots. Symptoms included shoot necrosis, stem browning, and wilted leaves. Shoots showing dieback symptoms were disinfected with 2% sodium hypochlorite, rinsed in sterile distilled water, and air dried. Five randomly selected fragments (5 × 5 mm) of infected shoots were cut and placed on petri dishes containing potato dextrose agar supplemented with ampicillin (100 μg/ml), and then incubated at 25°C in darkness. After 5 days, two whitish colonies were randomly isolated, coded as DUCC512 and DUCC513 for identification. When these isolates were cultured on oatmeal agar (OMA) at 25°C for 7 days in darkness, aerial mycelium was white and floccose to woolly. After 15 days of incubation, abundant black pycnidia were scattered over the medium. One hundred pycnidia and conidia from 15-day-old cultures were used to measure length and width. At 200× and 400× magnification, pycnidia were globose to subglobose and 40 to 110 × 42 to 150 μm. Conidia were aseptate, hyaline to brown, smooth walled, subglobose to oval, and 5 to 7 × 3 to 4.5 μm. These morphological properties correspond to Nothophoma quercina authentic strain CBS633.92 (Aveskamp et al. 2010; Chen et al. 2015). For molecular analysis, the partial β-tubulin gene was amplified from the genomic DNA of the isolates using the primer pair T10 (5′-CGATAGGTTCACCTCCAGAC-3′) and BT12 (5′-GTTGTCAATGCAGAAGGTCTCG-3′). Each of the partial β-tubulin nucleotide sequences was deposited in GenBank under accessions KX184308 and KX184309. BLAST analysis revealed that the β-tubulin sequences of the two isolates shared 100% identity (335/335) with that of N. quercina authentic strain (GU237609). On the basis of morphology and the sequences of β-tubulin gene, the isolates from necrotic lesions on C. sinensis were identified as N. quercina (Chen et al. 2015). The identified colonies were deposited in the Dankook University Culture Collection (DUCC). Pathogenicity tests were conducted on 5-month-old plants. After 7 days of incubation, a mycelial plug (0.3 cm2) cut from the margin of an actively growing colony of the fungus was placed into a shallow wound (0.3 cm2) infected with a sterilized scalpel on the stem base. Inoculated wounds were wrapped with Parafilm. For the control plants, sterile OMA plugs were separately placed into the artificial wounds of the stem base. Five replicates were made for each treatment and the plants were maintained in a greenhouse at 25°C. Six weeks after inoculation, inoculated trees reproduced stem browning and wilting symptoms, while control plants remained healthy. Koch’s postulates were satisfied after reisolating N. quercina from lesions on artificially inoculated stems. N. quercina has been reported to be responsible for stem canker from pistachio and olive trees in the United States and Tunisia (Chen et al. 2013; Taieb et al. 2014). To our knowledge, this is the first report of stem canker caused by N. quercina on C. sinensis in the Republic of Korea as well as other countries. This host plant is widely used for medicinal purpose in Asia. Therefore, our report would be useful information for the management of C. sinensis.

Ambrosia beetle Premnobius cavipennis (Scolytinae: Ipini) carries highly divergent ascomycotan ambrosia fungus, Afroraffaelea ambrosiae gen. nov. et sp. nov. (Ophiostomatales)

Abstract Ambrosia beetles and fungi represent an interesting and economically important symbiosis, but the vast majority of ambrosia fungi remain unexplored, hindering research, management of pathogens, and mitigation of invasive species. Beetles in the subtribe Premnobiini are one example of an entire beetle lineage whose fungal symbionts have never been studied. Here, we identify one dominant fungal symbiont of Premnobius cavipennis by using fungus culturing, community sequencing, microtome sectioning and micro-CT scanning of mycangia. Phylogenetic analyses of combined 18S and 28S rDNA and β-tubulin sequences revealed a highly divergent fungal lineage within Ophiostomatales, Afroraffaelea ambrosiae gen. nov. et sp. nov. The newly described fungal lineage represents another origin of the symbiosis within the Kingdom Fungi, adding to our understanding of the geographic ancestry of ambrosia fungi. P. cavipennis possesses pharyngeal mycangia which appear restrictive in fungus selection. This ambrosia beetle-fungus association has remained stable even after invasions into non-native regions.

Diversity in Secondary Metabolites Including Mycotoxins from Strains of Aspergillus Section Nigri Isolated from Raw Cashew Nuts from Benin, West Africa.

In a previous study, raw cashew kernels were assayed for the fungal contamination focusing on strains belonging to the genus Aspergillus and on aflatoxins producers. These samples showed high contamination with Aspergillus section Nigri species and absence of aflatoxins. To investigate the diversity of secondary metabolites, including mycotoxins, the species of A. section Nigri may produce and thus threaten to contaminate the raw cashew kernels, 150 strains were isolated from cashew samples and assayed for their production of secondary metabolites using liquid chromatography high resolution mass spectrometry (LC-HRMS). Seven species of black Aspergilli were isolated based on morphological and chemical identification: A. tubingensis (44%), A. niger (32%), A. brasiliensis (10%), A. carbonarius (8.7%), A. luchuensis (2.7%), A. aculeatus (2%) and A. aculeatinus (0.7%). From these, 45 metabolites and their isomers were identified. Aurasperone and pyranonigrin A, produced by all species excluding A. aculeatus and A. aculeatinus, were most prevalent and were encountered in 146 (97.3%) and 145 (95.7%) isolates, respectively. Three mycotoxins groups were detected: fumonisins (B2 and B4) (2.7%) ochratoxin A (13.3%), and secalonic acids (2%), indicating that these mycotoxins could occur in raw cashew nuts. Thirty strains of black Aspergilli were randomly sampled for verification of species identity based on sequences of β-tubulin and calmodulin genes. Among them, 27 isolates were positive to the primers used and 11 were identified as A. niger, 7 as A. tubingensis, 6 as A. carbonarius, 2 as A. luchuensis and 1 as A. welwitschiae confirming the species names as based on morphology and chemical features. These strains clustered in 5 clades in A. section Nigri. Chemical profile clustering also showed also 5 groups confirming the species specific metabolites production.

New Raffaelea species (Ophiostomatales) from the USA and Taiwan associated with ambrosia beetles and plant hosts.

Raffaelea (Ophiostomatales) is a genus of more than 20 ophiostomatoid fungi commonly occurring in symbioses with wood-boring ambrosia beetles. We examined ambrosia beetles and plant hosts in the USA and Taiwan for the presence of these mycosymbionts and found 22 isolates representing known and undescribed lineages in Raffaelea. From 28S rDNA and β-tubulin sequences, we generated a molecular phylogeny of Ophiostomatales and observed morphological features of seven cultures representing undescribed lineages in Raffaelea s. lat. From these analyses, we describe five new species in Raffaelea s. lat.: R. aguacate, R. campbellii, R. crossotarsa, R. cyclorhipidia, and R. xyleborina spp. nov. Our analyses also identified two plant-pathogenic species of Raffaelea associated with previously undocumented beetle hosts: (1) R. quercivora, the causative agent of Japanese oak wilt, from Cyclorhipidion ohnoi and Crossotarsus emancipatus in Taiwan, and (2) R. lauricola, the pathogen responsible for laurel wilt, from Ambrosiodmus lecontei in Florida. The results of this study show that Raffaelea and associated ophiostomatoid fungi have been poorly sampled and that future investigations on ambrosia beetle mycosymbionts should reveal a substantially increased diversity.

Identification of Colletotrichum spp. associated with fruit rot of Capsicum annuum in North Western Himalayan region of India using fungal DNA barcode markers

The DNA barcode approach was used to identify and establish association of Colletotrichum species complex with fruit rot disease of chili (Capsicum annuum L.) in North-Western Himalayan region of India. Twenty isolates of five morphologically identified Colletotrichum species collected from commercial chili growing areas were identified using deoxyribonucleic acid (DNA) barcode marker genes, 5.8S ribosomal ribonucleic acid flanking internal transcribed spacers 1 & 2 and β-tubulin gene. Morpho-cultural identification requires expertise to delineate C. gloeosporioides, C. boninense and C. acutatum complexes from each other, as these species possess minute variation in spore shape and size. Ribosomal DNA and β-tubulin sequence analysis along with species-specific marker amplification established the association of seven Colletorichum spp. viz., C. truncatum (syn. Colletotrichum capsici), C. coccodes, C. karstii, C. kahawae, C. nymphaeae, C. fructicola and C. gloeosporioides complex with fruit rot of chili. Phylogenetic analysis of 35 Colletotrichum sequences including authentic type sequences validated the identified sequences with strong bootstrap support. This approach delineated morphologically identified species with great ease into more reliable genotype based speciation of various Colletorichum complexes. The DNA barcode markers have direct implications for plant pathologists in relation to diagnostics in fields and for the purpose of quarantine and disease management.

Evaluation of matrix-assisted laser desorption/ionization time-of-fight mass spectrometry for identification of 345 clinical isolates of Aspergillus species from 11 Korean hospitals: comparison with molecular identification

We evaluated the ability of the Filamentous Fungi Library 1.0 of the MALDI-TOF MS Biotyper system to identify 345 clinical Aspergillus isolates from 11 Korean hospitals. Compared with results of the internal transcribed spacer region sequencing, the frequencies of correct identification at the species-complex level were 94.5% and 98.8% with cutoff values of 2.0 and 1.7, respectively. Compared with results of β-tubulin gene sequencing, the frequencies of correct identification at the species level were 96.0% (cutoff 2.0) and 100% (cutoff 1.7) for 303 Aspergillus isolates of five common, non-cryptic species, but only 4.8% (cutoff 1.7) and 0% (cutoff 2.0) for 42 Aspergillus isolates of six cryptic species (identifiable by β-tubulin or calmodulin sequencing). These results show that the MALDI Biotyper using the Filamentous Fungi Library version 1.0 enables reliable identification of the majority of common clinical Aspergillus isolates, although the database should be expanded to facilitate identification of cryptic species.

Two benzimidazole resistance-associated SNPs in the isotype-1 β-tubulin gene predominate in Haemonchus contortus populations from eight regions in China

Haemonchus contortus is one of the most important parasitic nematodes of small ruminants around the world, particularly in tropical and subtropical regions. The control of haemonchosis relies mainly on anthelmintics, but the excessive and prolonged use of anthelmintics is causing serious drug resistance issues in many countries. As benzimidazole (BZ) anthelmintics have been broadly used in China, we hypothesized that resistance is widespread. Given the link between three known single nucleotide polymorphisms (SNPs, designated F167Y, E198A and F200Y) in the isotype-1 β-tubulin gene and BZ resistance, our goal here was to explore the presence of these mutations in H. contortus from small ruminants (sheep and goats) from eight provinces in China using PCR-coupled sequencing. In addition, the genetic diversity and genetic relationship of isotype-1 β-tubulin sequence haplotypes were also investigated. Among 192 H. contortus adult individuals representing the eight populations, we identified six distinct sequence types, five of which had SNP E198A (GCA) and/or F200Y (TAC). Sequence analysis showed that the frequencies of SNPs E198A and F200Y were 0–70% and 0–31%, respectively. SNP F167Y (TAC) was not detected in any population. In addition, high haplotype diversities (0.455–0.939) and nucleotide diversities (0.018–0.039) were calculated. A network analysis of the isotype-1 β-tubulin gene sequences showed that SNPs E198A and F200Y occurred in multiple distinct groupings, suggesting multiple independent origins of these SNPs. The findings of this first study of SNPs in the isotype-1 β-tubulin gene of H. contortus populations suggest that BZ resistance is prevalent in some regions of China, and that any control strategy might focus on monitoring BZ resistance in this country.

Diversity and enzyme activity of Penicillium species associated with macroalgae in Jeju Island.

A total of 28 strains of 19 Penicillium species were isolated in a survey of extracellular enzyme-producing fungi from macroalgae along the coast of Jeju Island of Korea. Penicillium species were identified based on morphological and β-tubulin sequence analyses. In addition, the halo-tolerance and enzyme activity of all strains were evaluated. The diversity of Penicillium strains isolated from brown algae was higher than the diversity of strains isolated from green and red algae. The commonly isolated species were Penicillium antarcticum, P. bialowiezense, P. brevicompactum, P. crustosum, P. oxalicum, P. rubens, P. sumatrense, and P. terrigenum. While many strains showed endoglucanase, β-glucosidase, and protease activity, no alginase activity was detected. There was a positive correlation between halo-tolerance and endoglucanase activity within Penicillium species. Among 19 Penicillium species, three species-P. kongii, P. olsonii, and P. viticola-have not been previously recorded in Korea.

2-(m-Azidobenzoyl)taxol binds differentially to distinct β-tubulin isotypes

There are seven β-tubulin isotypes present in distinct quantities in mammalian cells of different origin. Altered expression of β-tubulin isotypes has been reported in cancer cell lines resistant to microtubule stabilizing agents (MSAs) and in human tumors resistant to Taxol. To study the relative binding affinities of MSAs, tubulin from different sources, with distinct β-tubulin isotype content, were specifically photolabeled with a tritium-labeled Taxol analog, 2-(m-azidobenzoyl)taxol, alone or in the presence of MSAs. The inhibitory effects elicited by these MSAs on photolabeling were distinct for β-tubulin from different sources. To determine the exact amount of drug that binds to different β-tubulin isotypes, bovine brain tubulin was photolabeled and the isotypes resolved by high-resolution isoelectrofocusing. All bands were analyzed by mass spectrometry following cyanogen bromide digestion, and the identity and relative quantity of each β-tubulin isotype determined. It was found that compared with other β-tubulin isotypes, βIII-tubulin bound the least amount of 2-(m-azidobenzoyl)taxol. Analysis of the sequences of β-tubulin near the Taxol binding site indicated that, in addition to the M-loop that is known to be involved in drug binding, the leucine cluster region of βIII-tubulin contains a unique residue, alanine, at 218, compared with other isotypes that contain threonine. Molecular dynamic simulations indicated that the frequency of Taxol-accommodating conformations decreased dramatically in the T218A variant, compared with other β-tubulins. Our results indicate that the difference in residue 218 in βIII-tubulin may be responsible for inhibition of drug binding to this isotype, which could influence downstream cellular events.

Diversity of Penicillium species isolated from heavy metal polluted soil in Guizhou Province, China

Seven Penicillium strains were isolated from soil samples polluted by heavy metals in different zones of Guizhou Province, China. Phenotypic identification proved to be difficult and this data was therefore supplemented with ribosomal internal transcribed spacer region and partial β-tubulin sequences. Comparison of the obtained sequences with references sequences showed that six strain belonged to Penicillium section Lanata-divaricata and one strain to section Sclerotiora. Three of the seven isolates belonged to a new species and were named here P. terrarumae sp. nov. The other four isolates were identified as P. cremeogriseum, P. guanacastense, P. griseopurpureum and P. oxalicum.

First Report of Neofusicoccum parvum Causing Shoot Blight and Stem Cankers on Pomegranate in Florida

HomePlant DiseaseVol. 100, No. 8First Report of Neofusicoccum parvum Causing Shoot Blight and Stem Cankers on Pomegranate in Florida PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Neofusicoccum parvum Causing Shoot Blight and Stem Cankers on Pomegranate in FloridaA. N. KC and G. E. ValladA. N. KCSearch for more papers by this author and G. E. ValladSearch for more papers by this authorAffiliationsAuthors and Affiliations A. N. KC G. E. Vallad , University of Florida, Gulf Coast Research and Education Center, Wimauma, FL 33598. Published Online:9 May 2016https://doi.org/10.1094/PDIS-01-16-0067-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat A survey on prevalence of pomegranate (Punica granatum L.) diseases in central Florida was conducted during the spring and summer of 2014 and 2015. Approximately 30% of the trees had blighted shoots and 10% had stem canker leading to death of entire tree. Symptomatic tissues were collected, surface-disinfected with 1% chlorine for 1 min, and placed on V8 juice agar amended with rifampicin (0.01 mg liter−1) and ampicillin (0.25 mg liter−1). After 3 days of incubation at 21°C and 12 h continuous light, fast-growing white colonies with abundant aerial mycelia were observed. The reverse sides of the plates were initially white but turned dark to olive green after 1 week of incubation. Observed conidiomata were globose and lined with conidiogenous cells. Conidia were hyaline, unicellular, ellipsoidal with flat base and round apex, and ranged from 10 to 14 × 3 to 5 µm (average of 50 conidia, 12 × 4 µm). The causal pathogen was identified as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (Crous et al. 2006; Palavouzis et al. 2015) based on morphological characteristics. For further confirmation, the rDNA internal transcribed spacer (ITS) region, translation elongation factor 1-α (EF-1α) gene, and β-tubulin gene were amplified on two isolates using the respective primer pairs ITS1 and ITS4, EF-AF and EF-BR, and T1 and T2, and subjected to Sanger sequencing. The sequence data were deposited in GenBank (Accession Nos. KU530156, KU530155, and KU530157 for ITS, EF-1α, and β-tubulin, respectively). A BLAST analysis with corresponding GenBank sequences found that the ITS, EF-1α, and β-tubulin sequences exhibited 100% (522/522), 99% (318/320), and 99% (603/605) identity with N. parvum (CMW37263 and GenBank KF432951; CBS 111524 and GenBank FJ150709; CCF216 and GenBank KC507808), respectively. Pathogenicity tests were conducted on three replicated sets of 2-year-old containerized trees by applying a 1 × 105 spores/ml conidial suspension of an N. parvum isolate with a handheld sprayer, and covering branches with plastic bags for 48 h to maintain high humidity. Three additional trees sprayed with sterile distilled water served as controls. Branches of inoculated trees started to blight 3 months after inoculation and exhibited abundant black conidiomata on the surface of infected stems similar to the symptoms and signs observed on stems in the field. After 6 months, cankers developed on the main stem. No symptoms developed on any control treatments. The fungus, N. parvum, was reisolated from all symptomatic tissues, satisfying Koch’s postulates. N. parvum has been reported to cause shoot blight of pomegranate in northern Greece, causing 5% yield loss with 2% disease incidence (Palavouzis et al. 2015). A similar species, N. mediterraneum, has been reported to cause cankers on the trunks and branches of pomegranate in California (Morgan and Michailides 2012). However, N. parvum is morphologically and phylogenetically distinct from N. mediterraneum (Crous et al. 2006). To our knowledge, this is the first report of N. parvum causing shoot blight and stem canker on pomegranate in Florida. The pathogen is of significant importance due to its ability to cause severe losses on pomegranate, an emerging alternative crop in southeastern United States.