Isozyme Variation among Biological Species in the Gibberella fujikuroi Species Complex (Fusarium Section Liseola)

Isozyme phenotypes were determined for 101 strains of Gibberella fujikuroi and 2 strains of Gibberella nygamai that represent seven biological species (mating populations) isolated from a variety of plant hosts in dispersed geographic locations. Fourteen enzymes were resolved in one or more of three buffer systems. Two of the enzymes, arylesterase and acid phosphatase, were polymorphic within two or more biological species and are suitable for intraspecific studies of population variation. Six enzymes, alcohol dehydrogenase, aspartate aminotransferase, glucose-6-phosphate dehydrogenase, mannitol dehydrogenase, phosphoglucomutase, and phosphogluconate dehydrogenase, were monomorphic in all of the isolates examined. The remaining six enzymes, fumarase, glucose phosphate isomerase, glutamate dehydrogenase (NADP), isocitrate dehydrogenase (NADP), malate dehydrogenase, and triose-phosphate isomerase, could potentially be used to distinguish the different biological species. Mating populations C and D are the most similar, since the mating population C isolates examined had the same isozyme phenotype as did a subset of the isolates in mating population D. Mating population E is the least similar to the other taxa examined. Unique isozyme phenotypes are present but are composed of banding patterns shared among the biological species. This finding supports the hypothesis that these biological species, with the possible exception of mating populations C and D, are reproductively isolated from one another and that no significant gene flow is occurring between them. Isozyme analysis is a useful method to distinguish these closely related biological species. Examination of isozyme phenotypes is more rapid than the present technique, which is based on sexual crosses; can be applied to strains that are not sexually fertile; and is more sensitive than traditional morphological characters, which cannot distinguish more than three or four morphological groups among the seven biological species. While emphasizing the discreteness of the mating populations as biological entities, our isozyme data also reaffirm the close genetic relationship among these groups.     

Development of PCR-RFLP Technique for Identify Several Members of Fusarium incarnatum-equiseti Species Complex and Fusarium fujikuroi Species Complex

Fusarium incarnatum-equiseti species complex (FIESC) contain over 40 members. The primer pair Smibo1FM/Semi1RM on the RPB2 partial gene has been reported to be able to identify Fusarium semitectum. The F. fujikuroi species complex (FFSC) contains more than 50 members. The F. verticillioides as a member of this complex can be identified by using VER1/VER2 primer pair on the CaM partial gene. In this research, the Smibo1FM/Semi1RM can amplify F. sulawesiense, F. hainanense, F. bubalinum, and F. tanahbumbuense, members of FIESC at 424 bp. The VER1/VER2 can amplify F. verticillioides, F. andiyazi, and F. pseudocircinatum, members of FFSC at 578 bp. Polymerase chain reaction-restriction fragment length polymorphism by using the combination of three restriction enzymes EcoRV, MspI, and HpyAV can differentiate each species of FIESC used. The two restriction enzymes HpaII and NspI can distinguish each species of FFSC used. The proper identification process is required for pathogen control in the field in order to reduce crop yield loss.     

Fusarium Species from the Fusarium fujikuroi Species Complex Involved in Mixed Infections of Maize in Northern Sinaloa,Mexico

Fusarium species belonging to the Fusarium fujikuroi species complex (FFSC) are associated with maize in northern Mexico and cause Fusarium ear and root rot. In order to assess the diversity of FFSC fungal species involved in this destructive disease in Sinaloa, Mexico, a collection of 108 fungal isolates was obtained from maize plants in 2007–2011. DNA sequence analysis of the calmodulin and elongation factor 1α genes identified four species: Fusarium verticillioides, F. nygamai, F. andiyazi and F. thapsinum (comprising 79, 23, 4 and 2 isolates, respectively). Differential distribution of Fusarium species in maize organs was observed, that is F. verticillioides was the most frequently isolated species from maize seeds, while F. nygamai predominated on maize roots. Mixed infections with F. verticillioides/F. thapsinum and F. verticillioides/F. nygamai were detected in maize seeds and roots, respectively. Pathogenicity assay demonstrated the ability of the four species to infect maize seedlings and induce different levels of disease severity, reflecting variation in aggressiveness, plant height and root biomass. Isolates of F. verticillioides and F. nygamai were the most aggressive. These species were able to colonize all root tissues, from the epidermis to the vascular vessels, while infection by F. andiyazi and F. thapsinum was restricted to the epidermis and adjacent cortical cells. This is the first report of F. nygamai, F. andiyazi and F. thapsinum infecting maize in Mexico and co‐infecting with F. verticillioides. Mixed infections should be taken into consideration due to the production and/or accumulation of diverse mycotoxins in maize grain.     

Mutual Exclusion between Fungal Species of the Fusarium Head Blight Complex in a Wheat Spike

Fusarium head blight (FHB) is one of the most damaging diseases of wheat. FHB is caused by a species complex that includes two genera of Ascomycetes: Microdochium and Fusarium. Fusarium graminearum, Fusarium culmorum, Fusarium poae, and Microdochium nivale are among the most common FHB species in Europe and were chosen for these experiments. Field studies and surveys show that two or more species often coexist within the same field or grain sample. In this study, we investigated the competitiveness of isolates of different species against isolates of F. graminearum at the scale of a single spike. By performing point inoculations of a single floret, we ensured that each species was able to establish independent infections and competed for spike colonization only. The fungal colonization was assessed in each spike by quantitative PCR. After establishing that the spike colonization was mainly downwards, we compared the relative colonization of each species in coinoculations. Classical analysis of variance suggested a competitive interaction but remained partly inconclusive because of a large between-spike variance. Further data exploration revealed a clear exclusion of one of the competing species and the complete absence of coexistence at the spike level.     

Molecular Phylogeny of Neotropical Anopheles (Nyssorhynchus) Albitarsis Species Complex (Diptera: Culicidae)

A phylogeny was reconstructed for four species belonging to the Neotropical Anopheles (Nyssorhynchus) albitarsis complex using partial sequences from the mitochondrial cytochrome oxidase I (COI) and NADH dehydrogenase 4 (ND4) genes and the ribosomal DNA ITS2 and D2 expansion region of the 28S subunit. The basis for initial characterization of each member of the complex was by correlated random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) markers. Analyses were carried out with and without an outgroup (An.(Nys.) argyritarsis Robineau-Desvoidy) by using maximum parsimony, maximum likelihood, and Bayesian methods. A total evidence approach without the outgroup, using separate models for "fast" (COI and ND4 position 3) and "slow" (rDNA ITS2 and D2, and COI and ND4 position 1) partitions, gave the best supported topology, showing close relationships of An. albitarsis Lynch-Arribálzaga to An. albitarsis B and An. marajoara Galv?o & Damasceno to An. deaneorum Rosa-Freitas. Analyses with the outgroup included showed poorer support, possibly because of a long branch attraction effect caused by a divergent outgroup, which caused one of the An. marajoara specimens to cluster with An. deaneorum in some analyses. The relationship of the above-mentioned result to a separately proposed hypothesis suggesting a fifth species in the complex is discussed.     

Genome-Wide Macrosynteny among Fusarium Species in the Gibberella fujikuroi Complex Revealed by Amplified Fragment Length Polymorphisms

The Gibberella fujikuroi complex includes many Fusarium species that cause significant losses in yield and quality of agricultural and forestry crops. Due to their economic importance, whole-genome sequence information has rapidly become available for species including Fusarium circinatum, Fusarium fujikuroi and Fusarium verticillioides, each of which represent one of the three main clades known in this complex. However, no previous studies have explored the genomic commonalities and differences among these fungi. In this study, a previously completed genetic linkage map for an interspecific cross between Fusarium temperatum and F. circinatum, together with genomic sequence data, was utilized to consider the level of synteny between the three Fusarium genomes. Regions that are homologous amongst the Fusarium genomes examined were identified using in silico and pyrosequenced amplified fragment length polymorphism (AFLP) fragment analyses. Homology was determined using BLAST analysis of the sequences, with 777 homologous regions aligned to F. fujikuroi and F. verticillioides. This also made it possible to assign the linkage groups from the interspecific cross to their corresponding chromosomes in F. verticillioides and F. fujikuroi, as well as to assign two previously unmapped supercontigs of F. verticillioides to probable chromosomal locations. We further found evidence of a reciprocal translocation between the distal ends of chromosome 8 and 11, which apparently originated before the divergence of F. circinatum and F. temperatum. Overall, a remarkable level of macrosynteny was observed among the three Fusarium genomes, when comparing AFLP fragments. This study not only demonstrates how in silico AFLPs can aid in the integration of a genetic linkage map to the physical genome, but it also highlights the benefits of using this tool to study genomic synteny and architecture.     

Osteological Characters to Define Six Species of the Capoeta damascina Species Complex (Cypriniformes: Cyprinidae)

Journal of Ichthyology - This paper presents the results of a comparative study of eight osteological characters: the counts, dimension and regionalization of the vertebral column, the pattern of...     

牵牛复合体（旋花科）的分类学研究

采用解剖镜和扫描电镜对牵牛复合体[牵牛Pharbitis nil（L．）Choisy、裂叶牵牛P.hederacea（L．）Choisy和圆叶牵牛P.purpurea（L．）Voigt]的叶形、萼片、果实和花粉形态进行了观察,同时对其叶绿体DNA trnL—F和核基因ITS进行了分析。结果表明,牵牛、裂叶牵牛与圆叶牵牛的外部形态,在叶形、萼片的形状及毛被、果实形态上的差异明显;花粉形态的差别也较为明显;在trnL-F和ITS序列分别构建的系统树中,牵牛、裂叶牵牛和圆叶牵牛聚为一个分支,牵牛和裂叶牵牛聚为一个小分支,ITS序列系统树的支持率为70％,trnL—F序列分子树的支持率仅为63％。根据以上不同,本研究认为牵牛、裂叶牵牛和圆叶牵牛都应作为种级处理。     

Distribution of Members of the Crypthecodinium cohnii (Dinophyceae) Species Complex1

Crypthecodinium cohnii, a small marine heterotrophic homothallic dinoflagellate, has diversified into a complex of morphologically very similar breeding groups (biological species or sibling species), some of which have become widely dispersed. Membership of two clones in the same sibling species is shown by their sexual compatibility as determined by genetic complementation in zygotes formed from motility mutants derived from the two stocks. Membership in different sibling species may be inferrec when motility mutants of one strain do not complement those of another. Fifty-six clones representing seaweed enrichments from *** geographic sites have been found to belong to 28 sibling species; 35 clones are members of seven wide-ranging biological species, and 21 are single representatives of 21 other breeding groups within the ranges of the others. Of 174 clonal isolates in our possession, 168 conform in size and shape to C. cohnii. Six others which have smaller cells and only one-fifth the standard DNA and chromosome number belong, we believe, to another species. The C. cohnii complex provides a unique opportunity for the study of evolutionary divergence and geographical dispersion of a dinoflagellate.     

Mycotoxin-producing Fusarium Species Occurring in Winter Wheat in Belgium (Flanders) During 2002–2005

Deoxynivalenol (DON) content and Fusarium spp. ( Fusarium graminearum , Fusarium culmorum , Fusarium avenaceum , Microdochium nivale and Fusarium poae ) of mycotoxin-producing Fusarium species in winter wheat in Belgium (Flanders) were determined. Field trials were set up in the varietal testing network of Flanders Agricultural Centre for Small Grains (Roeselare – Beitem, Belgium) and followed up during growing seasons 2001–2002, 2002–2003, 2003–2004 and 2004–2005. Fusarium infection and DON contamination were mainly influenced by location and environmental parameters. Mean DON levels ranged from 0 to 15 mg/kg. Over the period of four growing seasons cvs Deben, Ordeal and Napier had the highest DON contamination. Seasonal and local weather conditions before and during flowering together with local crop husbandry measures (crop rotation, soil preparation) seemed to be of great importance in explaining the variation in results obtained. At Bottelare a positive correlation between disease index and DON content was found for the growing seasons 2001–2002 and 2002–2003, but not the season 2003–2004. Fusarium graminearum and F. culmorum were in general the most frequently occurring Fusarium spp. in Flanders over the 4 years but the composition of the Fusarium population varied strongly from location to location and from year to year. Fusarium graminearum predominated in areas especially where maize was cultivated, whereas in areas with more small cereals in the crop rotation more F. culmorum was present. Also temperature played a role in the composition of Fusarium spp.     

The Model Organism Hermissenda crassicornis (Gastropoda: Heterobranchia) Is a Species Complex

Hermissenda crassicornis is a model organism used in various fields of research including neurology, ecology, pharmacology, and toxicology. In order to investigate the systematics of this species and the presence of cryptic species in H. crassicornis, we conducted a comprehensive molecular and morphological analysis of this species covering its entire range across the North Pacific Ocean. We determined that H. crassicornis constitutes a species complex of three distinct species. The name Hermissensa crassicornis is retained for the northeast Pacific species, occurring from Alaska to Northern California. The name H. opalescens is reinstated for a species occurring from the Sea of Cortez to Northern California. Finally, the name H. emurai is maintained for the northwestern species, found in Japan and in the Russian Far East. These three species have consistent morphological and color pattern differences that can be used for identification in the field.     

Electrophoretic Characterization of Members of the Crypthecodinium cohnii (Dinophyceae) Species Complex1

Eighty-seven axenic clones of the colorless inshore dinoflagellate Crypthecodinium cohnii were found by mating experiments to fall into 52 sibling species, seven wide ranging (two possibly global)—called major sibling species—and 45 found only once—minor sibling species. Electrophoretic analysis of three soluble enzymes from these strains revealed the following: 1) Despite some polymorphism most members of major sibling species closely resemble one another electrophoretically. 2) Major sibling species and most minor ones are electrophoretically distinct. 3) Sharing of electromorphs is sufficiently extensive, however, that no major sibling species is totally unrelated to all others. 4) Some minor sibling species are electrophoretically indistinguishable from a member of a major sibling species or from one another, suggesting recent origin by sexual isolation in situ. 5) Other minor sibling species differ from majors by one, two, or all three of the enzymes studied. A “model” of sexual isolation and diversification is offered.     

Species-Specific Detection and Identification of Fusarium Species Complex,the Causal Agent of Sugarcane Pokkah Boeng in China

Background

Pokkah boeng disease caused by the Fusarium species complex results in significant yield losses in sugarcane. Thus, the rapid and accurate detection and identification of the pathogen is urgently required to manage and prevent the spreading of sugarcane pokkah boeng.

Methods

A total of 101 isolates were recovered from the pokkah boeng samples collected from five major sugarcane production areas in China throughout 2012 and 2013. The causal pathogen was identified by morphological observation, pathogenicity test, and phylogenetic analysis based on the fungus-conserved rDNA-ITS. Species-specific TaqMan real-time PCR and conventional PCR methods were developed for rapid and accurate detection of the causal agent of sugarcane pokkah boeng. The specificity and sensitivity of PCR assay were also evaluated on a total of 84 isolates of Fusarium from China and several isolates from other fungal pathogens of Sporisorium scitamineum and Phoma sp. and sugarcane endophyte of Acremonium sp.

Result

Two Fusarium species (F. verticillioides and F. proliferatum) that caused sugarcane pokahh boeng were identified by morphological observation, pathogenicity test, and phylogenetic analysis. Species-specific TaqMan PCR and conventional PCR were designed and optimized to target their rDNA-ITS regions. The sensitivity of the TaqMan PCR was approximately 10 pg of fungal DNA input, which was 1,000-fold over conventional PCR, and successfully detected pokkah boeng in the field-grown sugarcane.

Conclusions/Significance

This study was the first to identify two species, F. verticillioides and F. proliferatum, that were causal pathogens of sugarcane pokkah boeng in China. It also described the development of a species-specific PCR assay to detect and confirm these pathogens in sugarcane plants from mainland China. This method will be very useful for a broad range of research endeavors as well as the regulatory response and management of sugarcane pokkah boeng.     

Molecular Evidence for Cryptic Speciation in the Cyclophorus fulguratus (Pfeiffer, 1854) Species Complex (Caenogastropoda: Cyclophoridae) with Description of New Species

A high degree of intraspecific variation, both genetic and in shell morphology, of the operculate land snail Cyclophorus fulguratus (Pfeiffer, 1854) suggests that its classification as a single species warrants reconsideration. We sequenced two nuclear (18S and 28S) and two mitochondrial (16S and COI) genes of 46 C. fulguratus specimens and used them to estimate the phylogeny and to determine the validity of species boundaries. Molecular phylogenetic analyses revealed the presence of three lineages corresponding to three geographically disjunctive populations of C. fulguratus in Thailand. Likelihood tests of topologies significantly supported the non-monophyly of the C. fulguratus–complex and Bayesian species delimitation analysis significantly supported the potential representation as distinct species of these three lineages. Discriminant function analysis based on geometric-morphometrics of shell shape allowed for significant distinction of these three candidate species, although they revealed a considerable degree of overlap of shell shape reflecting their crypsis morphologically. The diagnostic characters are provided by color pattern, pattern of protoconch and pattern of jaw. In conclusion, the results support that the C. fulguratus s.l., as currently recognized, consists of three distinct species in Thailand: C. fulguratus s.s., C. rangunensis and C. abditus sp.nov., which are described herein.     

Occurrence of Zearalenols (Diastereomeric Mixture) in Corn Stalk Rot and Their Production by Associated Fusarium Species

Zearalenol was extracted from Fusarium-infected stems of corn from southern Italy. The toxin, which appeared as a single compound in various thin-layer chromatography systems, was resolved by high-pressure liquid chromatography into two components. A gas chromatography-mass spectrometry examination of a purified fraction confirmed the natural occurrence of zearalenol as a diastereomeric mixture and led to the identification of alpha (56 ng/g) and beta (27 ng/g) isomers. Among nine Fusarium species found associated with stalk rot in corn, only Fusarium culmorum (F. roseum `Culmorum') and F. equiseti (F. roseum `Gibbosum') produced zearalenol and always produced it in a diastereomeric mixture of alpha and beta isomers.