Characterisation of members of the <Emphasis Type="Italic">Fusarium incarnatum</Emphasis>-<Emphasis Type="Italic">equiseti</Emphasis> species complex from undisturbed soils in South Africa

The genus Fusarium hosts a large number of economically significant phytopathogens with a global distribution. Surprisingly, only a limited number of studies have tried to identify the natural distribution of members of this genus in undisturbed soils. Members of the Fusarium incarnatum-equiseti species complex (FIESC) are increasingly associated with plant disease, and human and animal health problems. Recently, an outbreak of kikuyu poisoning of cattle was attributed to the F. incarnatum-equiseti species complex. Thus, it is of importance to identify the natural distribution of members of the FIESC from the environment. The aim of this study was to use the phylogenetic signal within the TEF 1α gene region to characterise 54 F. incarnatum-equiseti isolates obtained from undisturbed soils from the grassland biome of South Africa. These isolates were further compared with members of the FIESC previously associated with kikuyu poisoning of cattle. The phylogenetic analysis indicated a high level of variation within this species complex. Several members were closely related to isolates implicated in the death of cattle from infected kikuyu grass.     

Pathogenic ability and saline stress tolerance of two Fusarium isolates from Odontesthes bonariensis eggs

BackgroundSeveral fungal species represent a potential risk to embryos of Odontesthes bonariensis (Cuvier and Valenciennes, 1835), a euryhaline freshwater fish that lives in the Pampean inland waters and has potential economic relevance.AimsTo identify two fungi isolated from O. bonariensis eggs exposed to saline conditions and to characterize their pathogenicity and tolerance to sodium chloride solutions.MethodsThe isolates were identified by morphological features, and a preliminar phylogenetic analysis using sequences of translation elongation factor 1-alpha (EF-1α) and calmodulin (CAM) was performed. Koch's postulates were tested to identify the causative agent of fungal infection. The influence of NaCl on the fungal growth was evaluated in in vitro assays.ResultsThe isolates LPSC 1001 and 1002 were identified as representatives of the genus Fusarium, and belonging to the Fusarium incarnatum-Fusarium equiseti species complex (FIESC) and the Fusarium solani species complex (FSSC), respectively. Histological observations on eggs exposed in vitro to both isolates in infectivity assays confirmed the ability of the fungal isolates to penetrate to egg's chorionic membrane, leading to the death of embryos. Increasing NaCl concentration in the culture medium reduced the growth of the isolates LPSC 1001 and 1002, being completely inhibited at 160 and 120 g/l NaCl respectively.ConclusionsThe isolates LPSC 1001 (FIESC) and 1002 (FSSC) were identified as fungal pathogens to O. bonariensis eggs. The use of NaCl solutions as antifungal treatment was not effective to control the infection with these strains.     

Occurrence and variability of mycotoxigenicFusarium species associated to wheat and maize in the South West of Spain

The contamination of cereals with mycotoxins produced by species ofFusarium is an important risk to human and animal health. The toxigenic profile is different depending on theFusarium species considered and, in some species, differences can also be observed at intraspecific level. Information about the distribution and variability of the mycotoxigenicFusarium species allow prediction of the toxins that may occur and to devise control strategies. In this work, the occurrence of mycotoxigenicFusarium species associated to cereals was analysed in a wide sample of durum wheat fields (Triticum durum Desf.) and maize from the South West of Spain (Andalucía).F. equiseti, F. graminearum andF. culmorum were the most frequentFusarium species detected in wheat fields followed byF. sambucinum andF. avenaceum, whereas in the case of maize,F. verticillioides andF. proliferatum were the onlyFusarium species present. The relationships of the Spanish isolates from theF. equiseti, F. avenaceum andF. sambucinum species were analysed by nucleotide sequence comparison of a partial region of the Elongation Factor 1 alpha (EF-1α) with other sequences available in data bases. The results indicated thatF. avenaceum andF. equiseti showed high variability and that the SpanishF. equiseti isolates seemed to belong toF. equiseti type II. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: MCYT (AGL2004/07549/C05/5). M. Jurado was supported by pre-doctoral fellowship by the MCYT     

Molecular taxonomy of Dunaliella (Chlorophyceae), with a special focus on D. salina: ITS2 sequences revisited with an extensive geographical sampling

We used an ITS2 primary and secondary structure and Compensatory Base Changes (CBCs) analyses on new French and Spanish Dunallela salina strains to investigate their phylogenetic position and taxonomic status within the genus Dunaliella. Our analyses show a great diversity within D. salina (with only some clades not statistically supported) and reveal considerable genetic diversity and structure within Dunaliella, although the CBC analysis did not bolster the existence of different biological groups within this taxon. The ITS2 sequences of the new Spanish and French D. salina strains were very similar except for two of them: ITC5105 "Janubio" from Spain and ITC5119 from France. Although the Spanish one had a unique ITS2 sequence profile and the phylogenetic tree indicates that this strain can represent a new species, this hypothesis was not confirmed by CBCs, and clarification of its taxonomic status requires further investigation with new data. Overall, the use of CBCs to define species boundaries within Dunaliella was not conclusive in some cases, and the ITS2 region does not contain a geographical signal overall.     

Phylogenetic analysis of tetraploid wheat based on nuclear DMC1 gene

Tetraploid wheat (AABB or AAGG, 2n = 4x = 28) holds an important place in Triticum. It includes two allopolyploid species, Triticum turgidum and Triticum timopheevii. Many problems concerning the phylogenetic relationships among tetraploid wheat species remain unresolved. In this study, sequences data for the nuclear DMC1 gene from 61 accessions of Triticum and Aegilops species, representing diploid and tetraploid species, were used to examine the phylogenetic relationships among tetraploid wheat. Phylogenetic trees were constructed using maximum-likelihood and neighbor-joining approaches, and gene flow and genetic differentiation values were computed. The results indicated that the A genome of tetraploid wheat originated from T. urartu rather than T. monococcum, and Aegilops speltoides was the donor of the B and G genomes. Hulled tetraploid wheat accessions formed a subclade, and naked tetraploid wheat got other subclade, indicating that at least two intermediary subspecies were involved in the evolution of T. turgidum. Triticum turgidum and T. timopheevii might have simultaneously originated from a hybridization events. These results indicated that the DMC1 gene sequences are useful for resolution of the molecular phylogenetic relationships of tetraploid wheat.     

Fusarium incarnatum is associated with postharvest fruit rot of muskmelon (Cucumis melo)

Postharvest fruit rot was observed on muskmelon (Cucumis melo) on market shelves at a melon farm in the city of Hatyai, Songkhla, Thailand. Diseased muskmelon fruit displayed cotton-like mycelia on wounds and had brown to dark brown internal tissue. Based on morphological characteristics and molecular analysis of internal transcribed spacer (ITS) and translation elongation factor 1-α (TEF1-α) DNA sequences, the fungal pathogen was identified as Fusarium incarnatum. This species belongs to the Fusarium incarnatum-equiseti species complex (FIESC). A pathogenicity test was conducted to verify Koch's postulates, and F. incarnatum was observed to cause fruit rot on muskmelon; symptoms of the disease were similar to those seen in the field. However, only artificially wounded melons became infected, suggesting that F. incarnatum is an obligate wound-infecting pathogen. To our knowledge, this is the first report of F. incarnatum causing fruit rot of muskmelon in Thailand.     

Lycoperdon rupicola and L. subumbrinum: two new puffballs from Europe

Lycoperdon rupicola and L. subumbrinum are here described as new species. The former is a morphologically characteristic species forming small subglobose to pyriforme fruiting bodies with a prominent, protruding and stellately lobed stoma. In the phylogenetic analyses the six sequences of L. rupicola form a clade with 100% BS and 1.0 BPP, clustering with L. dermoxanthum. The proposed species grows among mosses on siliceous boulders and flat rocks as well as on open, sandy soil in temperate and hemiboreal regions. The records to date indicate its presence in Fennoscandia and Spain (Catalunya). L. subumbrinum morphologically resembles L. umbrinum but differs in having coarser ornamentation of its spores. In the phylogenetic analyses the five sequences of L. subumbrinum form a clade with 100% BS and 1.0 BPP, clustering with L. muscorum. The records to date, originating from southernmost Sweden and NW Slovakia, indicate that L. subumbrinum is a species of rich deciduous forests. L. muscorum is recorded for the first time from the Iberian peninsula (Spain).     

Polymorphism of mycotoxin biosynthetic genes among <Emphasis Type="Italic">Fusarium equiseti</Emphasis> isolates from Italy and Poland

Fusarium equiseti (Corda) Saccardo is a soil saprophyte and a weak pathogen, associated with several diseases of fruit and other crops in subtropical and tropical areas, but also in countries with temperate climate. A wide range of secondary metabolites has been identified among natural F. equiseti populations, with zearalenone (ZEA), fusarochromanone and fusarenon-X being the most common. In present study, the genetic diversity of strains from two populations (from Italy and Poland) was evaluated by analysing the translation elongation factor 1α (tef-1α) sequences, two polyketide synthases from the ZEA biosynthetic pathway (PKS13 and PKS4) and the TRI5 gene from the trichothecene biosynthetic pathway. ZEA was produced in rice cultures by 20 of the 27 tested isolates in concentrations ranging from 1.34 ng/g to 34,000 ng/g). The ability to produce enniatins and trichothecenes was evaluated in all strains by identifying esyn1, TRI13 and TRI4 genes. The presence of PKS4 and PKS13 genes was confirmed by polymerase chain reaction (PCR) in only some ZEA-producing isolates. Similarly, the TRI5 gene was found in 14 of the 27 isolates tested. This is likely to have been caused by the divergence of those genes between F. equiseti and F. graminearum (the latter species was used for the primers design) and can be exploited in phylogenetic studies. The analysis of the mycotoxin biosynthetic gene sequences can be used to differentiate the studied genotypes even more precisely than the analysis of the non-coding regions (like tef-1α).     

Regional Speciation or Taxonomic Inflation? The Status of Several Narrowly Distributed and Endangered Species of Narcissus Using ISSR and Nuclear Ribosomal ITS Markers

The mountains of the southeast Iberian Peninsula harbor several narrowly endemic species belonging to Narcissus section Pseudonarcissi (N. alcaracensis, N. bugei, N. enemeritoi, N. longispathus, N. nevadensis, N. segurensis, and N. yepesii) that are protected by regional Spanish laws. Most of these trumpet daffodils show a very similar overall morphology, and the correct identification of species is difficult unless the geographical origins of the accessions are known. ISSR (Inter-Simple Sequence Repeat) and nuclear ribosomal ITS sequences were used to assess patterns of genetic discontinuities present among the members of this section from southeast Spain. Species-specific ISSR bands were found in N. bugei, N. longispathus and N. nevadensis, but were absent in the other species. The multivariate ordination of the 288 ISSR-genotyped accessions showed that only N. longispathus and N. bugei were clearly differentiated, while the other species formed an unresolved group. ITS sequences of N. bugei were highly divergent from those retrieved from the remaining species, which were placed in a single largely unresolved clade in phylogenetic analysis. In conclusion, ISSR and ITS markers support the taxonomic distinction of N. bugei and N. longispathus. However, N. alcaracensis, N. segurensis, N. yepesii, N. enemeritoi, and N. nevadensis could be merged within a single species (N. nevadensis for nomenclatural priority).     

Phylogenetic analysis of the dimeric alpha-amylase inhibitor sequences from an orthologous region in 21 different genomes of the tribe Triticeae (Poaceae)

Six hundred and thirty gene sequences from 21 different genomes in Triticeae tribe were obtained and subjected to phylogenetic analysis. The sequences showed high homology in both nucleotide sequences and length variation, and had a common conserved cysteine skeleton C–Xn–C–Xn–C–Xn–CC–Xn–C–X–C–Xn–C–Xn–C–Xn–C. The sequences from common wheat formed three clusters; two were close to Aegilops tauschii and Aegilops speltoides sequences, respectively, and the third cluster was complex with sequences from Ae. speltoides, Aegilops searsii, and Aegilops bicornis. Different S genome(s) of Aegilops contributed α-amylase inhibitor loci to polyploid wheat by gene introgression in interspecific hybridizations. No sequence from common wheat was similar to that from einkorn wheat. We conclude that the occurrence of multiple chromosomal translocations or inversions in the different genomes of Triticeae had not dramatically affected the primary structure of dimeric α-amylase inhibitors. The results revealed important information on genome shaping events and processes occurring at the dimeric α-amylase inhibitor genes loci and their bearing on the phylogenetic relationships in the tribe Triticeae (Poaceae).     

Notes on the genus Polycoccum (Ascomycota,Dacampiaceae) in Spain,with a key to the species

Comments on and a key to the 13 Polycoccum species known in Spain are presented, including synopses of their world distributions. Amongst these is P. rubellianae sp. nov., a lichenicolous fungus growing on thalli of Caloplaca rubelliana in eastern Spain (Valencia). It has relatively small ascomata, the lower part pale brown, and also small ascospores which are coarsely verrucose and have a thick gelatinous sheath when young. The new species is associated with a Phoma-like anamorph. The identity and systematic position of P. opulentum requires further study as the name has been applied to different species, and the occurrence of P. marmoratum in Spain is in need of confirmation. The Spanish record of P. arnoldii on Lecania cyrtella probably refers to another yet undescribed species.     

Changes in duration of developmental phases of durum wheat caused by breeding in Spain and Italy during the 20th century and its impact on yield

Background and Aims

Although the apical development of wheat has been widely described, studies analysing how genetic breeding over the 20th century influenced the developmental phases and its consequences on yield generation are lacking, especially for durum wheat under field conditions in Mediterranean environments. The aims of this study were to analyse the effects of breeding in Spain and Italy on crop development during the last century, to determine whether or not breeding significantly altered the developmental phases between sowing and maturity, and to evaluate the importance of each phase in determining the number of grains per spike of durum wheat (Triticum durum) cultivars representing the germplasm grown throughout the 20th century in Spain and Italy.

Methods

Eight field experiments were carried out during 4 years in two contrasting latitudes (Lleida and Granada, Spain). Plant material consisted of 24 durum wheat cultivars (12 Italian and 12 Spanish) grown throughout the 20th century in Spain and Italy.

Key Results

In Spanish materials, breeding reduced the duration of the period from sowing to anthesis, placing the grain-filling period in better conditions. In those cultivars, the sub-phase sowing–terminal spikelet formation was reduced while the duration of the period from booting to anthesis was increased. The number of grains per spike increased by 23 % from old to modern cultivars, by changes in the number of grains per spikelet in both Spanish and Italian cultivars. Floral abortion from booting to anthesis diminished by 24 % from old to modern cultivars, and grain setting increased by 13 %.

Conclusions

The results suggest that breeding reduced not only plant height, but also the time to anthesis. By extending the duration of the phase from booting to anthesis, which was associated with an increase in spike dry weight and grains per spike, it suggests that future increases in spike fertility could be achieved by enlarging that phase.     

Entosthodon mouretii (Corb.) Jelenc new to Europe

Entosthodon mouretii (Corb.) Jelenc, a species related to E. fascicularis (Hedw.) C. Müll., is referred for the first time in Europe, from samples collected in Spain. The Spanish specimens are described and illustrated.     

Molecular characterization and phylogenetic analysis of Wx genes from three Taeniatherum diploid species

In wheat seeds, starch synthase I or the Waxy protein is an enzyme involved in amylose synthesis. The gene encoding this enzyme is Wx and in this study, eight novel Wx alleles were identified in three diploid Taeniatherum species. The variability of these alleles was evaluated, and their nucleotide sequences were compared with those of homologous alleles from wheat. Two types of Taeniatherum Wx alleles were detected in three diploid species Ta. caput-medusae, Ta. asperum, and Ta. crinitum. A phylogenetic analysis indicates that the Taeniatherum Wx alleles were more closely related to Wx alleles from Aegilops species with C, D, M, and U genomes than to Wx alleles of other species. These alleles represent a potential genetic resource that may be useful in wheat breeding programs.     

Subterranean termite phylogeography reveals multiple postglacial colonization events in southwestern Europe

A long‐standing goal of evolutionary biology is to understand how paleoclimatic and geological events shape the geographical distribution and genetic structure within and among species. Using a diverse set of markers (cuticular hydrocarbons, mitochondrial and nuclear gene sequences, microsatellite loci), we studied Reticulitermes grassei and R. banyulensis, two closely related termite species in southwestern Europe. We sought to clarify the current genetic structure of populations that formed following postglacial dispersal from refugia in southern Spain and characterize the gene flow between the two lineages over the last several million years. Each marker type separately provided a fragmented picture of the evolutionary history at different timescales. Chemical analyses of cuticular hydrocarbons and phylogenetic analyses of mitochondrial and nuclear genes showed clear separation between the species, suggesting they diverged following vicariance events in the Late Miocene. However, the presence of intermediate chemical profiles and mtDNA introgression in some Spanish colonies suggests ongoing gene flow. The current genetic structure of Iberian populations is consistent with alternating isolation and dispersal events during Quaternary glacial periods. Analyses of population genetic structure revealed postglacial colonization routes from southern Spain to France, where populations underwent strong genetic bottlenecks after traversing the Pyrenees resulting in parapatric speciation.     

Genetic diversity of Fasciola hepatica in Spain and Peru

In the present study, molecular characterization of Fasciola flukes from Spain was performed to reveal the relation with the previously reported Peruvian F. hepatica population. The nuclear DNA markers, phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold), were used for species identification of Fasciola flukes. A total of 196 Fasciola flukes were identified as F. hepatica by pepck and pold, and 26 haplotypes were detected in mitochondrial NADH dehydrogenase subunit 1 (nad1). Only one of them was previously found in Spanish samples; which indicates the existence of high genetic diversity and population structure in F. hepatica from Spain. Three haplotypes were identical to those from Peruvian F. hepatica. The pairwise fixation index value confirmed a relatively close relationship between the Spanish and Peruvian F. hepatica samples. The Spanish samples showed clearly higher genetic variability than the Peruvian population. These results are discussed in relation with the hypothesis of the introduction of the parasite in America from Europe and recent evidence of pre-Hispanic F. hepatica from Argentina revealed by ancient DNA.     

Waxy genes from spelt wheat: new alleles for modern wheat breeding and new phylogenetic inferences about the origin of this species

Background and Aims

Waxy proteins are responsible for amylose synthesis in wheat seeds, being encoded by three waxy genes (Wx-A1, Wx-B1 and Wx-D1) in hexaploid wheat. In addition to their role in starch quality, waxy loci have been used to study the phylogeny of wheat. The origin of European spelt (Triticum aestivum ssp. spelta) is not clear. This study compared waxy gene sequences of a Spanish spelt collection with their homologous genes in emmer (T. turgidum ssp. dicoccum), durum (T. turgidum ssp. durum) and common wheat (T. aestivum ssp. aestivum), together with other Asian and European spelt that could be used to determine the origin of European spelt.

Methods

waxy genes were amplified and sequenced. Geneious Pro software, DNAsp and MEGA5 were used for sequence, nucleotide diversity and phylogenetic analysis, respectively.

Key Results

Three, four and three new alleles were described for the Wx-A1, Wx-B1 and Wx-D1 loci, respectively. Spelt accessions were classified into two groups based on the variation in Wx-B1, which suggests that there were two different origins for the emmer wheat that has been found to be part of the spelt genetic make-up. One of these groups was only detected in Iberian material. No differences were found between the rest of the European spelt and the Asiatic spelt, which suggested that the Iberian material had a different origin from the other spelt sources.

Conclusions

The results suggested that the waxy gene variability present in wheat is undervalued. The evaluation of this variability has permitted the detection of ten new waxy alleles that could affect starch quality and thus could be used in modern wheat breeding. In addition, two different classes of Wx-B1 were detected that could be used for evaluating the phylogenetic relationships and the origins of different types of wheat.     

Detection of a novel Chlamydia species in captive spur-thighed tortoises (Testudo graeca) in southeastern Spain and proposal of Candidatus Chlamydia testudinis

The spur-thighed tortoise (Testudo graeca) is an endangered Mediterranean tortoise that lives in North Africa, Southern Europe and Southwest Asia. In the wake of recent legislation making their keeping as domestic animals illegal, many of these animals have been returned to wildlife recovery centers in Spain. In the present study, a population of such tortoises showing signs of ocular disease and nasal discharge was examined for the presence of Chlamydia spp. Cloacal, conjunctival and/or choanal swabs were collected from 58 animals. Using a real-time PCR specific for the family Chlamydiaceae, 57/58 animals tested positive in at least one sample. While only a few samples proved positive for C. pecorum, sequencing of the 16S rRNA gene revealed a sequence identical to previously published sequences from specimens of German and Polish tortoises. Whole-genome sequences obtained from two conjunctival swab samples, as well as ANIb, TETRA values and a scheme based on 9 taxonomic marker genes revealed that the strain present in the Spanish tortoises represented a new yet non-classified species, with C. pecorum being its closest relative. We propose to designate the new species Candidatus Chlamydia testudinis.     

Identification and Pathogenicity of Fungal Pathogens Causing Black Point in Wheat on the North China Plain

Fungi associated with black point were isolated from three highly susceptible wheat genotypes in the North China Plain. The 21 isolates represented 11 fungal genera. The most prevalent genera were Alternaria (isolation frequency of 56.7%), Bipolaris (16.1%), and Fusarium (6.0%). The other eight genera were Curvularia, Aspergillus, Cladosporium, Exserohilum, Epicoccum, Nigrospora, Penicillium, and Ulocladium; their isolation frequencies ranged from 0.8 to 4.8%. The pathogenicity of the isolates was individually assessed in the greenhouse by inoculating wheat plants with spore suspensions. Ten of the 21 isolates caused significantly higher incidences of black point than that the controls. These isolates belonged to eight fungal species (A. alternata, B. sorokiniana, B. crotonis, B. cynodontis, C. spicifera, F. equiseti, E. rostratum, and E. sorghinum) based on morphological traits and phylogenetic analysis. The average incidences of black point in the eight fungal species were 32.4, 54.3, 43.0, 41.9, 37.2, 38.8, 50.1, and 34.1%, respectively. B. sorokiniana and A. alternata were determined to be the most important pathogens in the North China Plain based on fungal prevalence and symptom severity. This study is the first to identify E. rostratum as a major pathogen causing black point in wheat.