Ribosomal DNA sequence polymorphism and the delineation of two ascosporic yeast species: Metschnikowia agaves and Starmerella bombicola

The relationship between mating success and sequence divergence in the internal transcribed spacer (ITS)/5.8S-D1/D2 rDNA region was examined in isolates tentatively assigned to Metschnikowia agaves and Starmerella bombicola. Both species are haplontic and heterothallic, such that the formation of mature asci can be used as a measure of genetic compatibility. Parsimony haplotype network analysis and mating success confirmed that all known isolates of M. agaves are conspecific. The previously reported D1/D2 polymorphism of five substitutions was not corroborated; the maximum divergence observed between any two strains was three substitutions, four with ITS. Of 39 putative S. bombicola strains, 36 formed an ITS-D1/D2 haplotype network using the 95% criterion. Thirty-five strains could mate with one or more compatible partner. The excluded strains did not mate. Mature asci arose from crosses between individuals differing by as many as five, but not six or seven substitutions in the D1/D2 domain. All strains capable of mating formed mature asci with at least one partner and all network members could be linked to another member by three or fewer substitutions. These results support the use of sequence divergence as a criterion for species delineation, but caution against describing poorly sampled species solely on the basis of that criterion.     

围小丛壳Glomerella cingulata子囊孢子交配繁殖、生物学特性及致病性

炭疽叶枯病（Glomerella leaf spot）是我国苹果上新发现的一种病害。为了解围小丛壳Glomerella cingulata子囊孢子的交配方式、生物学特性和致病性,从安徽砀山、山东牟平等地采集病害样品,经分离培养和纯化获得单孢菌株。在适宜条件下单孢菌株可产生子囊和子囊孢子,经过毛细管破子囊壁后单孢分离,获得12个子囊,每个子囊有8个子囊孢子。其中10个子囊中有4个“正”孢子（+）和4个“负”孢子（-）,2个子囊中只有“负”孢子。子囊孢子单孢菌株培养72h,“正”菌株菌落白色,以营养生长为主;“负”菌株菌落灰白色,直径略小于正菌株,菌丝稀疏,边缘菌丝白色,中部有大量橙色的分生孢子堆。“正”、“负”菌株异宗配合后,可产生大量可育子囊壳;单独的“正”菌株有性生殖产生稀疏丛簇状的可育子囊壳;单个的“负”菌株只能产生分散且不育的子囊壳。“正”、“负”菌株菌落的生长速度没有差异,对温度、营养、光照和pH值的敏感性也没有差异,但“正”、“负”菌株的致病性存在差异。正菌株的有性生殖没有导致rDNA-ITS、β-tubulin基因碱基序列变异。     

Occurrence of Gibberella zeae strains that produce both nivalenol and deoxynivalenol

By single ascospore isolation, several sets of asci containing eight ascospores were isolated from perithecia of Gibberella zeae. Of these sets, seven were investigated for their ability to produce 8-ketotrichothecene mycotoxins on rice grains. Analyses were made with gas chromatography-mass spectrometry and gas chromatography with 63Ni electron capture detection. Of 56 total isolates, 11 produced nivalenol, 4-acetylnivalenol, and deoxynivalenol, 1 produced nivalenol and deoxynivalenol, 7 produced deoxynivalenol and 3-acetyldeoxynivalenol, 19 produced deoxynivalenol and 15-acetyldeoxynivalenol, and 6 produced deoxynivalenol and both 15- and 3-acetyldeoxynivalenol. The remaining 12 isolates produced nivalenol and 4-acetylnivalenol. All isolates of G. zeae that we examined could produce 8-ketotrichothecenes in this investigation. This report is the first to demonstrate the presence of G. zeae isolates producing both nivalenol and deoxynivalenol. In addition, differences in the production between 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol are discussed in relation to culture conditions.     

Occurrence of Gibberella zeae strains that produce both nivalenol and deoxynivalenol.

By single ascospore isolation, several sets of asci containing eight ascospores were isolated from perithecia of Gibberella zeae. Of these sets, seven were investigated for their ability to produce 8-ketotrichothecene mycotoxins on rice grains. Analyses were made with gas chromatography-mass spectrometry and gas chromatography with 63Ni electron capture detection. Of 56 total isolates, 11 produced nivalenol, 4-acetylnivalenol, and deoxynivalenol, 1 produced nivalenol and deoxynivalenol, 7 produced deoxynivalenol and 3-acetyldeoxynivalenol, 19 produced deoxynivalenol and 15-acetyldeoxynivalenol, and 6 produced deoxynivalenol and both 15- and 3-acetyldeoxynivalenol. The remaining 12 isolates produced nivalenol and 4-acetylnivalenol. All isolates of G. zeae that we examined could produce 8-ketotrichothecenes in this investigation. This report is the first to demonstrate the presence of G. zeae isolates producing both nivalenol and deoxynivalenol. In addition, differences in the production between 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol are discussed in relation to culture conditions.     

Aquatic fungi from peat swamp palms: Phruensis brunneispora gen. et sp. nov. and its hyphomycete anamorph

Phruensis brunneispora is a new genus and species occurring on decaying trunks of the palm Licuala longecalycata in Sirindhorn Peat Swamp Forest, Thailand. We compare the genus with other aquatic ascomycetes with falcate septate ascospores: Pseudohalonectria and Ophioceras. Ascospores differ from species in these genera in being brown with lighter end cells. Also, the ascus pore is subapical, with a channel leading to the apex. Lollipopaia minuta differs from Phruensis brunneispora in that the ascomata are borne in a stroma, asci have an apical pore and the ascospores are hyaline. No genus was found to accommodate the new species. Molecular analysis of rDNA ribosomal 18S confirmed the exclusion of the new species from Pseudohalonectria, and Ophioceras and Lollipopaia minuta formed a sister group with it. Phruensis brunneispora and Lollipopaia minuta grouped in the Diaporthales with 100% bootstrap support. Therefore, both morphological and molecular evidence supports erecting a new genus to accommodate this taxon. A hyaline Phialophora-like anamorph was formed when single ascospores were plated out on agar. The taxon is described and illustrated with light micrographs.     

江苏省稻瘟病菌有性态的研究*

用标准菌株对１９９７～１９９９年在江苏省吴江市,宜兴市、通州市、高邮市和赣榆县采集的３２５个猪瘟病菌单孢分离菌株的可育性和交配型进行了测定,结果表明江苏省稻瘟病菌菌株的育性较低,可交配率为２２．７７％,可育率仅为７．０８％。不同年份、不同地区采集的猪瘟病菌茵株的性亲和力和交配型有较大的差异,三年的交配率分别为２６．６１％、８．２６％和３３．６４％;温州地区和赣榆地区菌株的交配率相对较高,分别为２６．１５％和２５．４２％,宜兴地区菌株的交配率较低,只有１５．３８％。江苏省稻瘟病菌菌株的交配型在不同年份亦出现很大差别,１９９７年２９个可交配菌株中有２１个菌株表现为ＭＡＴ１－２ 交配型,而１９９９年３６个可交配茵株均为ＭＡＴ１－１交配型。用江苏省稻瘟病菌的可育菌株进行互交,２５个组合中只有６个组合能产生子囊壳和子囊,但均不产生子囊孢子,提示江苏省稻瘟病菌在田间产生健康有性后代的几率不大。对杂交后代的遗传学分析表明,菌株的交配型是受单基因控制的。     

Fusarium subglutinans f. sp. Pini represents a distinct mating population in the gibberella fujikuroi species complex

Fusarium strains in the Gibberella fujikuroi species complex cause diseases on a variety of economically important plants. One of these diseases, pitch canker of Pinus spp., is caused by strains identified as Fusarium subglutinans f. sp. pini. Fertile crosses were detected between F. subglutinans f. sp. pini strains from South Africa, California, and Florida. F. subglutinans f. sp. pini strains were not cross-fertile with the standard tester strains of six of the seven other mating populations of G. fujikuroi. Sporadic perithecia with ascospores were obtained in two crosses with the mating population B tester strains. These perithecia were homothallic, and the ascospores derived from these perithecia were vegetatively compatible with the mating population B tester strain parent. We concluded that fertile F. subglutinans f. sp. pini isolates represent a new mating population (mating population H) of G. fujikuroi and that they belong to a unique biological species in a distinct taxon.     

EFFECT OF CARBON AND NITROGEN NUTRITION ON REPRODUCTION IN LEPTOSPHAERULINA BRIOSIANA

Reproduction of Leptosphaerulina briosiana was studied on modified Richard's medium supplemented with seven carbon compounds, six amino acids, five inorganic nitrogen compounds or with urea. The fungus grew on each of the media, but it did not reproduce on all. Ascostromata formed on each of the carbon sources, but the formation of asci and ascospores and the ejection of ascospores varied with the carbon source and the isolate of the fungus. Ascostromata formed on all the nitrogen sources except arginine and ammonium sulfate. Formation of asci and ascospores and ejection of the ascospores varied with the nitrogen source and the isolate of the fungus. The fungus grew and produced ascostromata on Richard's medium supplemented with each of six vitamins, but asci and ascospores were not formed.     

A new sterigmatocystin-producing Emericella variant from agricultural desert soils

An unusual, sterigmatocystin-producing taxon with characteristics of both Emericella nidulans (anamorph Aspergillus nidulans) and Emericella rugulosa (anamorph Aspergillus rugulovalvus, formerly A. rugulosus) was isolated repeatedly during a mycofloral survey of desert cotton field soils where aflatoxin is a chronic problem. Members of this taxon had ascospores with smooth convex walls like E. nidulans but grew slowly like E. rugulosa; moreover, they were similar to an industrial echinocandin B-producing strain which had been classified as "Aspergillus nidulans var. roseus." These new desert isolates were compared with "A. nidulans var. roseus" and representative wild-type isolates of E. nidulans and E. rugulosa using traditional morphological characters, secondary metabolite profiles of mycelial extracts, and Southern blot analysis of genomic DNA. The desert isolates and "A. nidulans var. roseus shared morphological, physiological and molecular characters with E. rugulosa. These isolates constitute a new non-rugulose variant of E. rugulosa.     

Chaenothecopsis quintralis, a new species of calicioid fungus

Chaenothecopsis quintralis from southwestern Argentina is described and illustrated as a new species in the family Mycocaliciaceae. It has been found in three localities in the Andean Patagonian temperate forests, growing strictly on dung of an endemic marsupial Dromiciops gliroides. The new species is distinguished by the hemispherical, black capitulum of ascoma, the presence of asci with croziers, one-celled brown ascospores, and its fimicolous habitat. Analysis of partial nuclear large subunit rDNA (LSU) sequences showed that this taxon is within Mycocaliciales.     

Dyrithiopsis lakefuxianensis gen. et sp. nov. from Fuxian Lake, Yunnan, China, and notes on the taxonomic confusion surrounding Dyrithium

A new taxon with Dyrithium-like characteristics was collected from Lake Fuxian in China. The taxon is typical of the Amphisphaeriaceae in that it has relatively large, ostiolate, immersed ascomata, unitunicate asci with a J+ subapical ring, and brown ascospores. It is similar to Dyrithium in that it has muriform ascospores, but considerable confusion surrounds this genus. In Dyrithium asci are bitunicate and lack a J+ subapical ring, while this was not true of our species. A new genus, Dyrithiopsis, therefore is established to accommodate this new taxon. Details of its anamorph also are provided, based on cultural studies. Parsimony analyses of part of the large-subunit rDNA provide further evidence to support the familial placement of this new genus in the Amphisphaeriaceae. The taxonomic position of Dyrithium also is discussed.     

Expression of Meiotic Drive Elements Spore Killer-2 and Spore Killer-3 in Asci of Neurospora Tetrasperma

It was shown previously that when a chromosomal Spore killer factor is heterozygous in Neurospora species with eight-spored asci, the four sensitive ascospores in each ascus die and the four survivors are all killers. Sk-2K and Sk-3K are nonrecombining haplotypes that segregate with the centromere of linkage group III. No killing occurs when either one of these killers is homozygous, but each is sensitive to killing by the other in crosses of Sk-2K x Sk-3K. In the present study, Sk-2K and Sk-3K were transferred by recurrent backcrosses from the eight-spored species Neurospora crassa into Neurospora tetrasperma, a pseudohomothallic species which normally makes asci with four large spores, each heterokaryotic for mating type and for any other centromere-linked genes that are heterozygous in the cross. The action of Sk-2K and Sk-3K in N. tetrasperma is that predicted from their behavior in eight-spored species. A sensitive nucleus is protected from killing if it is enclosed in the same ascospore with a killer nucleus. Crosses of Sk-2K x Sk-2S, Sk-3K x Sk-3S, and Sk-sK x Sk-3K all produce four-spored asci that are wild type in appearance, with the ascospores heterokaryotic and viable. The Eight-spore gene E, which shows variable penetrance, was used to obtain N. tetrasperma asci in which two to eight spores are small and homokaryotic. When killer and sensitive alleles are segregating in the presence of E, only those ascospores that contain a killer allele survive. Half of the small ascospores are killed. In crosses of Sk-2K x Sk-3K (with E heterozygous), effectively all small ascospores are killed. The ability of N. tetrasperma to carry killer elements in cryptic condition suggests a possible role for Spore killers in the origin of pseudohomothallism, with adoption of the four-spored mode restoring ascospore viability of crosses in which killing would otherwise occur.     

Conlarium duplumascospora gen. et. sp. nov. and Jobellisia guangdongensis sp. nov. from freshwater habitats in China

Conlarium duplumascospora gen. et. sp. nov. and Jobellisia guangdongensis sp. nov. are described and illustrated from submerged wood collected from Guangdong Province, China. Conlarium duplumascospora is characterized by gregarious, coriaceous and beaked ascomata; cylindrical, unitunicate asci with a bipartite apical ring; biseriate, fusiform, hyaline, 0-5-septate ascospores with or without appendages; and anamorph with muriform conidia. Jobellisia guangdongensis is characterized by globose to subglobose, gregarious and papillate ascomata; three-layered peridium; cylindrical, unitunicate asci with a refractive apical ring; and one-septate, fusiform, greenish brown ascospores. Sequence analyses of partial nuclear large subunit ribosomal DNA (LSU rDNA) were performed to infer the phylogenetic affinities of these new taxa. A key to species of Jobellisia is provided.     

A new species of Epichloë symbiotic with Chinese grasses

Epichlo? species are fungal symbionts (endophytes) of grasses, six European and four North American biological species in genus Epichlo? have been described in previous researches. In this study we describe a new Epichlo? species, Epichlo? yangzii Li et Wang, found in natural symbioses with Roegneria kamoji native to China. We investigated the host specificity, morphology, interfertility tests and molecular phylogenetic evidences of this new species. The results indicated that E. yangzii is host specific and seedborne. Most morphological characteristics of this new species are typical in the genus. However differences are evident in several features including size of perithecia, asci and ascospores. In mating tests E. yangzii was not interfertile with E. elymi isolates from related hosts in genera Elymus. Phylogenetic relationships based on sequences of beta-tubulin gene (tub2) introns and translation elongation factor 1-alpha gene (tef1) introns showed that members of the new species grouped into exclusive clades with high bootstrap value.     

Chaenothecopsis khayensis, a new resinicolous calicioid fungus on African mahogany

The new species Chaenothecopsis khayensis (Ascomycota, Mycocaliciaceae) is described from Ghana, western Africa, on the resin of Khaya anthotheca and K. ivorensis. The species is distinctive in forming asci without crosiers and in possessing ascospores that are faintly longitudinally striate. Analysis of large subunit rDNA gene sequences positioned this species within a clade corresponding to the Mycocaliciales and identified its closest relative as Sphinctrina leucopoda. Chaenothecopsis khayensis occurs commonly on resin exuding from trees damaged by the larvae of the mahogany shoot borer (Hypsipyla sp.), and we discuss the possible ecological relationship between the fungus and these moths.     

Clavispora,a new yeast genus of the Saccharomycetales

In Candida lusitaniae van Uden et do Carmo-Sousa (1959), strains of opposite sex have been found. Cells of the opposite mating types conjugate and form asci with one to four clavate spores. These are easily liberated from the ascus. The type strain of Candida obtusa (Dietrichson) van Uden et do Carmo-Sousa ex van Uden et Buckley (1970) also produces ascospores after mating with one of the strains of Candida lusitaniae. As clavate ascospores are unknow in yeasts, a new genus, Clavispora, is proposed with Clavispora lusitaniae spec. nov. as type species.     

Incubation of excised apothecia enhances ascus maturation of Sclerotinia sclerotiorum

Synchronized maturation of ascospores of Sclerotinia sclerotiorum is desirable for establishing a transformation system, conducting genetic analyses of the pathogen, defining the precise epidemiological roles of ascospores and screening plant germplasm for resistance. In general, fresh apothecia collected from germinated sclerotia contained primarily immature or discharged asci. This study was undertaken to investigate whether maturation of asci and ascospores could be enhanced by incubation of excised apothecia and to determine the effects of factors such as temperature, excision time, light and ventilation on maturation of asci and ascospores in excised apothecia. Maturation of asci was compared between intact and excised apothecia that were incubated under similar conditions. Results demonstrated that temperature was an important factor affecting ascus maturation of S. sclerotiorum during incubation of excised apothecia, and the optimum temperature was around 21 C. After incubation at 21 C for 30 h, the percentage of undischarged mature asci in excised apothecia increased up to 70-80%. This increase was accompanied by a significant increase in ascospore production of up to 5 x 10(5) ascospores per apothecium. Detailed time course studies indicated that mature asci peaked at 30-36 h of postexcision incubation. Mature asci and the number of ascospores were higher in open incubation than in closed incubation, suggesting that accumulation of volatile substances was not required for ascus/ascospore maturation during postexcision incubation and ventilation could enhance the maturation process. Light also did not affect the maturation of asci during the incubation of excised apothecia. Germination rates for ascospores from excised apothecia under various treatments were similar to those from untreated apothecia but declined slightly with time postexcision. The incubation of excised apothecia promoted ascus maturation compared with intact apothecia.     

我国北方地区裸胞壳属(Emericela)的种

对１４０２份来自于东北、华北及西北地区的土样进行裸胞壳属（Ｅｍｅｒｉｃｅｌａ）菌的分离。结合菌落形态及扫描电镜下子囊孢子的形态将各菌鉴定到种。结果表明我国北方地区分布有８个种,１个变种,分别是无冠裸胞壳（Ｅｍｅｒｉｃｅｌａａｃｒｉｓｔａｔａ）、皱折裸胞壳（４Ｅｍｅｒｉｃｅｌａｃｏｒｒｕｇａｔａ）、茴香裸胞壳（Ｅｍｅｒｉｃｅｌａｆｏｅｎｉｃｕｌｉｃｏｌａ）、宫治裸胞壳（Ｅｍｅｒｉｃｅｌａｍｉｙａｊｉ）、构巢裸胞壳（Ｅｍｅｒｉｃｅｌａｎｉｄｕｌａｎｓｖａｒ．ｎｉｄｕｌａｎｓ）、构巢裸胞壳宽脊变种（Ｅｍｅｒｉｃｅｌａｎｉｄｕｌａｎｓｖａｒ．ｌａｔａ）、四脊裸胞壳（Ｅｍｅｒｉｃｅｌａｑｕａｄｒｉｌｉｎｅａｔａ）、褶皱裸胞壳（Ｅｍｅｒｉｃｅｌａｒｕｇｕｌｏｓａ）及波状裸胞壳（Ｅｍｅｒｉｃｅｌａｕｎｄｕｌａｔａ）。除银川地区以Ｅｍｅｒｉｃｅｌａａｃｒｉｓｔａｔａ为优势菌外,各地区以Ｅｍｅｒｉｃｅｌａｎｉｄｕｌａｎｓ最为常见。文中对常见菌种进行了简要的概述,对在我国较少见的种进行了详细的描述和讨论     

Neurospora discreta,a new heterothallic species defined by its crossing behavior

The species is described and namedNeurospora discreta sp. nov. because of its stringent reproductive isolation. Isolates collected from burned vegetation at a single site near Kirbyville, Texas, include both mating types (Aanda). Experimental criteria based on cross-fertility were used for assigning species status. Crosses between isolates of opposite mating type are highly fertile, producing abundant eightspored asci. In contrast, when the Kirbyville strains are crossed to sexually compatible speciestester strains representingN. crassa, N. intermedia, N. sitophila, andN. tetrasperma, perithecia are rudimentary and no ascospores are produced. The haploid chromosome number is 7. Chromosomes at pachytene resemble those of otherNeurospora species. Biotin is required. Linear growth is slower than for other heterothallic species. When A and a strains from Kirbyville grow toward one another and intersect on crossing medium, there is no barrage. A single homogeneous band of perithecia is formed where they meet, indicating that opposite mating types are vegetatively compatible. The Kirbyville population differs from other heterothallicNeurospora species in ascospore morphology and vegetative traits. Ascospores from Kirbyville parents are larger, and the ribs between confluent parallel grooves are ornamented with dot-like pits. Vegetative cultures from Kirbyville are yellowish rather than orange, and large empty barren protoperithecia or false perithecia are produced abundantly in unfertilized haploid cultures. Isolates from two otherN. discreta populations resemble otherNeurospora species more closely with respect to these morphological traits but are clearly conspecific with the Kirbyville strains on the basis of fertility in crosses.