Translation Elongation Factor 1-alpha Sequences Research Articles

Morosphaeria muthupetensis sp. nov. (Morosphaeriaceae) from India: morphological characterization and multigene phylogenetic inference

Abstract A novel species of Morosphaeria, Morosphaeria muthupetensis, saprobic on intertidal mangrove wood of Rhizophora mucronata from Muthupet mangroves on Kaveri River Delta, Tamil Nadu along the East coast of India, is described in this paper. Morosphaeria muthupetensis is characterized by large sub-globose to lenticular ascomata; long cylindrical to clavate, short pedicellate asci; small, fusiform to ellipsoidal ascospores with acute ends when compared to other Morosphaeria species. A multigene phylogeny obtained from a combined nuclear large subunit, small subunit, internal transcribed spacers rDNA and translation elongation factor 1-alpha sequence analysis revealed that M. muthupetensis is phylogenetically distinct and nested in between Morosphaeria velatispora and Morosphaeria ramunculicola and all these species constitute a strongly supported monophyletic clade sister to the genus Helicascus. Detailed descriptions and illustrations of M. muthupetensis are presented and compared with other Morosphaeria species.

First Report of Fusarium avenaceum Causing Postharvest Decay on ‘Conference’ Pears in the Netherlands

Pear (Pyrus communis) is an important fruit crop in the Netherlands, with a total production of 349,000 tons in 2014. ‘Conference’ is the main pear cultivar that occupies 75% of the total pear production area in the Netherlands. In the Netherlands, pears are kept in controlled atmosphere cold storage for 9 to 11 months after harvest. Lesions were observed on pears of the cultivar Conference in a survey carried out from 2012 to 2014 in packing houses in the Netherlands. In general, low incidences of 1 to 5% were recorded. Lesions showed brown and watery circular necrosis, were slightly sunken, often with visible whitish, yellowish, or pink mycelia covering the lesions. Fruits were rinsed with sterile water, and lesions were sprayed with 70% ethanol until droplet runoff. The skin was removed aseptically with a scalpel, and tissue under the lesion was isolated and placed onto potato dextrose agar (PDA). The PDA plates were incubated at 20°C in the dark, and single spore isolates were propagated on fresh PDA plates. These isolates produced fast-growing colonies with extensive aerial mycelium which was initially white, then turning yellow to pink and a dark pink to red reverse. Macroconidia were slightly falcate, thin-walled, usually 5 septate, with a tapering apical cell, and measured 40 to 80 × 3.5 to 5 μm. Both cultural and morphological characteristics of the pathogen were similar to those described for Fusarium spp. The identity of two representative isolates (Fu5-44261 and Fu6-44280) from different pear lots was confirmed by means of multilocus gene sequencing. Genomic DNA was extracted using the LGC Mag Plant Kit (Berlin) in combination with the Kingfisher method (Waltham, MA). Sequences of ITS region, translation elongation factor 1-alpha (TEF1), and histone H3 (HIS3) loci were amplified and sequenced. The sequences have been deposited in GenBank under accession numbers KT350588 and 89 (ITS), KT350605 and 06 (TEF1), and KT935569 and 70 (HIS3). MegaBLAST analysis revealed that our ITS sequences matched with 99.8 to 100% identity to Fusarium spp. belonging to the Fusarium tricinctum species complex. The TEF1 sequences of both isolates were 99 to 100% identical with F. avenaceum culture collection sequences in GenBank (JQ429374 and GQ915502). The HIS3 sequences of both isolates were 97 to 99% identical with the sequences of accessions JQ435857 and GQ915469, confirming the identity of these isolates as F. avenaceum. Koch’s postulates were performed on 15 ‘Conference’ pears per isolate. Surface sterilized fruit were inoculated with 20 μl of a suspension of 105 macroconidia ml–1 prepared from a 15-day-old PDA culture, after wounding with a needle. Inoculated fruits were sealed in a plastic bag and incubated in darkness at 20°C. Symptoms appeared after 4 to 6 days on 100% of the fruits while mock-inoculated controls with water remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA morphologically resembled the original isolate from the infected pears. Symptoms observed on artificially inoculated ‘Conference’ pear fruit were identical to the decay observed on ‘Conference’ pears that were obtained from cold storage. Only few reports describe symptoms associated with F. avenaceum on apple in Europe and the United States (Kou et al. 2014; Sever et al. 2012; Sorensen et al. 2009), while F. avenaceum infections of pears have not been reported. Thus, this is the first report of storage decay caused by F. avenaceum on pear. F. avenaceum infections may constitute a safety issue due to the potential production of mycotoxins such as moniliformin (Sorensen et al. 2009).

First Report of Neonectria candida Causing Postharvest Decay on ‘Conference’ Pears in the Netherlands

Pear (Pyrus communis) is an important fruit crop in the Netherlands, with a total production of 349,000 tons in 2014, and ‘Conference’ is the main pear cultivar that occupies 75% of the total pear production area. In the Netherlands, pears are kept in controlled atmosphere cold storage up to 11 months after harvest. Occasionally, storage rots are observed when storage crates are contaminated with orchard soil. In a storage trial (2012 to 2013), boxes with ‘Conference’ pears were amended with soil particles from the same orchard from which the pears were harvested (four orchards), and stored for 11 months. Boxes without amended soil were included as controls. In contrast to the control boxes, up to 15% of the pears stored in boxes with soil particles showed typical rot symptoms (lesions) of an unknown causal agent. The lesions showed brown and watery circular necrosis, were slightly sunken, and displayed whitish to yellowish mycelia covering the lesions. To isolate the causal agent, fruit were rinsed with sterile water, lesions were sprayed with 70% ethanol until droplet runoff, the skin was removed aseptically with a scalpel, and tissue under the lesion was isolated and placed onto potato dextrose agar (PDA). The PDA plates were incubated at 20°C in the dark, and single spore isolates were transferred to fresh PDA plates. These isolates produced fast-growing colonies with white-yellowish mycelium. Conidia were hyaline, cylindrical, 1 to 3 septate, and 15.8 to 26.4 × 5.3 to 7.9 µm. The fungus was morphologically identical to Neonectria candida (syn. N. ramulariae; anamorph Cylindrocarpon obtusiusculum) (Lombard et al. 2015). The identity of a representative isolate (VTN10Bs3) was confirmed by means of multilocus gene sequencing. To this end, genomic DNA was extracted using the LGC Mag Plant Kit (Berlin, Germany) in combination with the Kingfisher method (Waltham, USA). Sequences of the ITS region, translation elongation factor 1-alpha (TEF1), and actin (ACT2) loci were amplified, sequenced, and deposited in GenBank under accessions KU588183 (ITS), KU588186 (TEF1), and KU588184 (ACT2). MegaBLAST analysis revealed that our ITS, TEF1, and ACT2 sequences matched with >99 to 100% identity to N. candida isolates in GenBank (KM249079 and JF735314 [ITS], JF735791 and HM054091 [TEF1], and KM231146 [ACT2]). Subsequently, Koch’s postulates were performed on 15 ‘Conference’ pears. Surface sterilized fruits were inoculated with 20 µl of a suspension of 105 conidiospores ml–1 water, prepared from a 15-day-old PDA culture, after wounding with a needle. Inoculated fruits were sealed in a plastic bag and incubated in darkness at 20°C. Symptoms appeared after 7 days on 100% of the fruits while mock-inoculated controls with water remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA morphologically resembled the original isolate from the infected pears. Moreover, symptoms observed on artificially inoculated ‘Conference’ pear fruit were identical to the decay observed on ‘Conference’ pears that were obtained from the cold storage experiment. The identity of the reisolations was confirmed as N. candida by sequencing. N. candida (syn. N. ramulariae) is known as a globally distributed soilborne fungus (Domsch et al. 2007), but only few studies have identified the fungus as plant pathogen (Hirooka 2012). This is the first report of N. candida causing storage rot of pears. Importantly, we note that the occurrence of storage rots may be enhanced by contamination of storage crates or fruit with orchard soil.

Two new Lycoperdon species collected from Korea: L. albiperidium and L. subperlatum spp. nov.

Thirty-four Lycoperdon specimens from Korea were examined with the internal transcribed spacer (ITS) region of ribosomal DNA sequence data. The result of the ITS sequences phylogenetic analysis indicated that the Korean specimens represented nine different species. To confirm the taxonomic position of these species, we conducted an intensive morphological investigation, and additional phylogenetic investigation of the protein coding regions RNA polymerase subunit II (RPB2) and translation elongation factor 1-alpha (TEF1). We discovered two new species (L. albiperidium and L. subperlatum) and one (L. ericaeum) newly discovered in Korea. Lycoperdon albiperidium is closely related to L. ericaeum based on ITS, RPB2 and TEF1 sequence data, but these species were distinguishable by morphological characteristics, especially the shape of the basidiocarps, the diameter of the eucapillitial threads and the size of the basidospores. Lycoperdon subperlatum is quite similar to the European and American L. perlatum based on morphological characteristics. However, L. subperlatum is clearly distinct from European and American L. perlatum based on ITS, RPB2 and TEF1 sequence data, and somewhat differs from them in macro- and microscopic characteristics. Based on morphological characteristics, L. ericaeum is related to L. subumbrinum and L. lividum but it is distinguishable by the presence of fragile, eucapillitial threads, the diameters of the threads and ITS sequences. Here, we describe four Lycoperdon species collected in Korea.

First Report of Fusarium subglutinans Causing Leaf Spot Disease on Cymbidium Orchids in Korea

In 2006~2010, leaf spot symptoms, that is, small, yellow spots that turned into dark brown-to-black lesions surrounded by a yellow halo, were observed on Cymbidium spp. in Gongju, Taean, and Gapyeong in Korea. A Fusarium species was continuously isolated from symptomatic leaves; in pathogenicity testing, isolates caused leaf spot symptoms consisting of sunken, dark brown lesions similar to the original ones. The causal pathogen was identified as Fusarium subglutinans based on morphological and translation elongation factor 1-alpha sequence analyses. This is the first report of F. subglutinans as the cause ofleaf spot disease in Cymbidium spp. in Korea.

Phylogeny and taxonomy of species in the Grosmannia serpens complex

Grosmannia serpens was first described from pine in Italy in 1936 and it has been recorded subsequently from many countries in both the northern and southern hemispheres. The fungus is vectored primarily by root-infesting bark beetles and has been reported to contribute to pine-root diseases in Italy and South Africa. The objective of this study was to consider the identity of a global collection of isolates not previously available and using DNA sequence-based comparisons not previously applied to most of these isolates. Phylogenetic analyses of the ITS2-LSU, actin, beta-tubulin, calmodulin and translation elongation factor-1 alpha sequences revealed that these morphologically similar isolates represent a complex of five cryptic species. Grosmannia serpens sensu stricto thus is redefined and comprises only isolates from Italy including the ex-type isolate. The ex-type isolate of Verticicladiella alacris was shown to be distinct from G. serpens, and a new holomorphic species, G. alacris, is described. The teleomorph state of G. alacris was obtained through mating studies in the laboratory, confirming that this species is heterothallic. Most of the available isolates, including those from South Africa, USA, France, Portugal and some from Spain, represent G. alacris. The remaining three taxa, known only in their anamorph states, are described as the new species Leptographium gibbsii for isolates from the UK, L. yamaokae for isolates from Japan and L. castellanum for isolates from Spain and the Dominican Republic.

First report of Fusarium redolens from Saskatchewan and its comparative pathogenicity

Fusarium redolens [syn: F. oxysporum var. redolens] is pathogenic on a wide range of plant species. Fusarium redolens was frequently isolated from necrotic and discolored root and crown tissues of chickpea, pea, lentil and durum wheat in Saskatchewan. The fungus was identified using distance analysis of the translation elongation factor-1 alpha sequences. Comparative pathogenicity tests showed that F. redolens, like Fusarium graminearum and Cochliobolus sativus, caused lesions and/or discolorations on the root, crown and shoot of durum wheat, pea and chickpea, although it had a different host preference. While F. redolens was most virulent on pea, the other two fungi were more virulent on durum wheat. It appears that F. redolens is common in the Prairie Ecozone of Saskatchewan and it may cause yield reduction in pea, chickpea and durum crops. This warrants a closer look at the biology and ecology of F. redolens in the Canadian Prairies.

First Detection of Penicillium fellutanum from Stored Rice in Korea

A representative isolate KU53 of the predominant Penicillium species was obtained from rice samples from rice processing complexes of National Agricultural Cooperative Federation in Korea. In this study, isolate KU53 was identified by its morphological and molecular characteristics. The macro- and microscopic characteristics of isolate KU53 were compared with the P. fellutanum reference isolate KCTC16913 on different media; isolate KU53 was generally identical to those of the reference isolate KCTC16913. In a molecular-based identification, the <TEX>${\beta}$</TEX>-tubulin and translation elongation factor 1-alpha sequences of isolate KU53 was most closely related to those of P. fellutanum. Thus, isolate KU53 from stored rice could be identified as P. fellutanum, some isolates of which are known to produce mycotoxin-related metabolites. To our knowledge, this is the first detection of P. fellutanum from stored rice in Korea.

Positive selection inAvrP4avirulence gene homologues across the genusMelampsora

Pathogen genes involved in interactions with their plant hosts are expected to evolve under positive Darwinian selection or balancing selection. In this study a single copy avirulence gene, AvrP4, in the plant pathogen Melampsora lini, was used to investigate the evolution of such a gene across species. Partial translation elongation factor 1-alpha sequences were obtained to establish phylogenetic relationships among the Melampsora species. We amplified AvrP4 homologues from species pathogenic on hosts from different plant families and orders, across the inferred phylogeny. Translations of the AvrP4 sequences revealed a predicted signal peptide and towards the C-terminus of the protein, six identically spaced cysteines were identified in all sequences. Maximum likelihood analysis of synonymous versus non-synonymous substitution rates indicated that positive selection played a role in the evolution of the gene during the diversification of the genus. Fourteen codons under significant positive selection reside in the C-terminal 28 amino acid region, suggesting that this region interacts with host molecules in most sequenced accessions. Selection pressures on the gene may be either due to the pathogenicity or avirulence function of the gene or both.

Trichoderma martiale sp. nov., a new endophyte from sapwood of Theobroma cacao with a potential for biological control

The new species Trichoderma martiale was isolated as an endophyte from sapwood in trunks of Theobroma cacao (cacao, Malvaceae) in Brazil. Based on sequences of translation-elongation factor 1-alpha ( tef1) and RNA polymerase II subunit ( rpb2) T. martiale is a close relative of, and morphologically similar to, T. viride, but differs in the production of discrete pustules on corn meal–dextrose agar (CMD) and SNA, in having a faster rate of growth, and in being a tropical endophyte. This new species was shown, in small-scale, in situ field assays, to limit black pod rot of cacao caused by Phytophthora palmivora, the cause of black pod disease.