Difference in antifungal activity of morphotypes of clavicipitaceous endophytes within and between species

In a previous study, a total of 484 endophytic fungi were isolated and purified from seven populations of Achnatherum sibiricum (L.) Keng collected at six geographical locations in Inner Mongolia, China. Based on growth rates as well as morphological characteristics, the isolates were classified into five morphotypes. Among them, morphotypes A, B and C were ascribed to the same species, Neotyphodium chisosum, based on ITS sequences. Morphotype E was identified as Epichlo? amarillans. In the present study, four morphotypes, A, B, C and E, belonging to two species, were chosen for an in vitro pathogen trial. The results showed that both endophyte colonies and endophyte filtrate of all morphotypes could inhibit the mycelia growth and spore germination of the pathogen fungi tested. The magnitude of inhibition varied not only between species, but also among morphotypes of the same species. Overall, the antifungal ability of E. amarillans (morphotype E) was higher than that of N. chisosum. Within N. chisosum, the antifungal ability was highest in morphotype C, followed by morphotype A, and lowest in morphotype B. This variability suggests that different morphotypes might represent different genotypes of endophyte. The effect of endophyte infection on the host grass should be examined not only on the species level but also on the morphotype level to determine the possible interactions.     

内蒙古中东部草原羽茅内生真菌的分类

从内蒙古中东部草原7个不同地理种群的羽茅Achnatherum sibiricum中分离、培养得到484株内生真菌,根据其形态学特征进行了分类学鉴定。结果表明,其中大部分菌株属于Neotyphodium内生真菌,并发现有少量Neotyphodium有性型Epichloё菌株存在。通过与已发表模式菌株的比较,鉴定其中两个种为N.chisosum和N.huerfanum。N.chisosum为羽茅内生真菌的优势种,而N.huerfanum是首次在羽茅中被发现。对其余暂未能确定种的菌株的菌落和显微形态特征进行了详细描述。不同地理种群羽茅内生真菌的分离率有自东向西增加的趋势,而内生真菌的形态多样性有自东向西减少的趋势。     

Difference in antifungal activity of morphotypes of clavicipitaceous endophytes within and between species

In a previous study, a total of 484 endophytic fungi were isolated and purified from seven populations of Achnatherum sibiricum (L.) Keng collected at six geographical locations in Inner Mongolia, China. Based on growth rates as well as morphological characteristics, the isolates were classified into five morphotypes. Among them, morphotypes A, B and C were ascribed to the same species, Neotyphodium chisosum, based on ITS sequences. Morphotype E was identified as Epichloë amarillans. In the present study, four morphotypes, A, B, C and E, belonging to two species, were chosen for an in vitro pathogen trial. The results showed that both endophyte colonies and endophyte filtrate of all morphotypes could inhibit the mycelia growth and spore germination of the pathogen fungi tested. The magnitude of inhibition varied not only between species, but also among morphotypes of the same species. Overall, the antifungal ability of E. amarillans (morphotype E) was higher than that of N. chisosum. Within N. chisosum, the antifungal ability was highest in morphotype C, followed by morphotype A, and lowest in morphotype B. This variability suggests that different morphotypes might represent different genotypes of endophyte. The effect of endophyte infection on the host grass should be examined not only on the species level but also on the morphotype level to determine the possible interactions.     

The evolutionary origins of three new Neotyphodium endophyte species from grasses indigenous to the Southern Hemisphere

Members of the genus Neotyphodium are asexual, seedborne, protective fungal endophytes of cool season grasses that have likely evolved either directly from sexual Epichlo?; species, or by the interspecific hybridization of distinct lineages of Epichlo?; and Neotyphodium. We investigated the evolutionary origins of Neotyphodium endophytes from several grasses that are indigenous to the Southern Hemisphere using a multiple-gene phylogenetic approach. Intron regions of the genes encoding β-tubulin (tub2), translation elongation factor 1-α (tef1) and actin (act1) were amplified by polymerase chain reaction and sequenced. Phylogenetic analyses of these sequences, aligned with homologous sequences from Epichlo?; spp., revealed the evolutionary origins of the Southern Hemisphere endophytes, where one lineage of apparently non-hybrid origin, and three lineages of unique interspecific hybrid origin were identified. On the basis of morphology, host range and evolutionary history, we propose three new species of Neotyphodium. Neotyphodium aotearoae was isolated from Echinopogon ovatus populations from New Zealand and Australia, and comprised a unique, apparently non-hybrid lineage within the Epichlo?; species phylogeny. In contrast, an interspecific hybrid lineage was identified from two Australian Ec. ovatus populations, whose ancestry apparently involved lineages closely related to extant E. festucae and an E. typhina genotype similar to that of isolates from Poa pratensis. Endophytes infecting South African Melica racemosa and M. decumbens (dronkgras) appeared to be hybrids of E. festucae and N. aotearoae or close relatives. The names N. australiense and N. melicicola are proposed for these two hybrid lineages, respectively. The origin of N. tembladerae, an established endophyte species from South American Poa and Festuca spp., was also investigated. Neotyphodium tembladerae appeared to be of hybrid origin, involving E. festucae and an E. typhina genotype similar to that of isolates from Poa nemoralis. The results of this study highlight the widespread occurrence of interspecific hybrid Neotyphodium lineages on a global scale, and the extent of endophyte gene-flow between the Northern and Southern Hemispheres.     

不同传播方式的内生真菌感染对羽茅的生理生态影响北大核心CSCD

通过田间试验,研究了垂直传播的Neotyphodium属内生真菌和水平传播的Epichloё属内生真菌对羽茅(Achnatherum sibiricum)生理生态的影响。结果表明:Neotyphodium属内生真菌感染对羽茅的株高和叶长有显著的正效应,而Epichloё属内生真菌感染对羽茅株高和叶长的增益作用较少。感染Neotyphodium属内生真菌的羽茅最大净光合速率显著高于感染Epichloё属内生真菌的羽茅,Neotyphodium属内生真菌显著提升了羽茅的气孔限制值和水分利用效率,而实验测定的第一年结果显示:Epichloё属内生真菌对羽茅的各个光合指标具有明显的负效应(胞间CO2浓度除外)。Neotyphodium属内生真菌感染使得宿主植物积累的可溶性糖含量显著高于感染Epichloё属内生真菌的羽茅和不染菌的羽茅植株。感染Epichloё属内生真菌的羽茅中氮含量显著高于感染Neotyphodium属内生真菌的羽茅。总之,内生真菌的传播方式是影响羽茅生理生态学特性的一个因素,并且垂直传播的内生真菌对羽茅生长和生理特性的益处高于水平传播的内生真菌。     

内蒙古中东部草原羽茅内生真菌的遗传多样性

该文从形态学和基因水平上对内蒙古中东部草原4个羽茅(Achnatherum sibiricum)种群所含的27个内生真菌(Neotyphodium)菌株的遗传多样性进行了研究,结果表明羽茅内生真菌在形态学和基因水平上均表现出较高的遗传多样性。在PDA培养基上,根据菌落颜色、质地、生长速率和分生孢子外观等特征,可分为4个形态型;西乌旗、霍林郭勒和定位站3个种群所含的内生真菌菌落具有相似的生长特性和外观,而羊草(Leymus chinensis)样地种群无论从菌落外观和质地的多样性,还是遗传多样性指数均高于其它3个样地。用20个随机引物对这27株内生真菌基因组DNA进行RAPD分析,共检测到463个位点,其中461个为多态位点,多态位点百分率达99.6%,特有位点93个,占20.1%。Nei基因多样性指数h和Shannon多样性指数I分别为 0.238 0和0.387 0;多数菌株间的遗传一致度较低而遗传距离较大,在DNA水平上存在较显著的遗传变异。基于Nei无偏遗传距离和UPGMA法的聚类分析结果将27株内生真菌分为7组,并且与形态型的划分基本一致,RAPD分析方法提高了内生真菌类群划分的可靠性。     

Species boundaries in gregarine apicomplexan parasites: a case study-comparison of morphometric and molecular variability in Lecudina cf. tuzetae (Eugregarinorida, Lecudinidae)

Trophozoites of gregarine apicomplexans are large feeding cells with diverse morphologies that have played a prominent role in gregarine systematics. The range of variability in trophozoite shapes and sizes can be very high even within a single species depending on developmental stages and host environmental conditions; this makes the delimitation of different species of gregarines based on morphological criteria alone very difficult. Accordingly, comparisons of morphological variability and molecular variability in gregarines are necessary to provide a pragmatic framework for establishing species boundaries within this diverse and poorly understood group of parasites. We investigated the morphological and molecular variability present in the gregarine Lecudina cf. tuzetae from the intestines of Nereis vexillosa (Polychaeta) collected in two different locations in Canada. Three distinct morphotypes of trophozoites were identified and the small subunit (SSU) rDNA was sequenced either from multicell isolates of the same morphotype or from single cells. The aim of this investigation was to determine whether the different morphotypes and localities reflected phylogenetic relatedness as inferred from the SSU rDNA sequence data. Phylogenetic analyses of the SSU rDNA demonstrated that the new sequences did not cluster according to morphotype or locality and instead were intermingled within a strongly supported clade. A comparison of 1,657 bp from 45 new sequences demonstrated divergences between 0% and 3.9%. These data suggest that it is necessary to acquire both morphological and molecular data in order to effectively delimit the "clouds" of variation associated with each gregarine species and to unambiguously reidentify these species in the future.     

醉马草内生菌的分离、鉴定及杀虫研究

摘要：【目的】探明醉马草内生菌的种类,筛选对农作物虫害有毒杀作用的菌株。【方法】采用研磨法从健康醉马草植物的根、茎、叶和种子中进行菌种分离;通过对其形态、培养特征、生理生化及其他生物学特性的研究;16S rDNA及ITS序列的系统发育学分析进行鉴定;用玻片浸渍法和喷雾法筛选产杀虫活性物质菌株。【结果】获得细菌89株,分别属于枯草芽孢杆菌属（Bacillus）、链霉菌属(Streptomyces)、棒状杆菌属（Corynebacterium）、叶杆菌属（Phyllobacterium）、鞘脂单胞菌属（Sph     

Simple sequence repeat markers distinguish among morphotypes of Sphaeropsis sapinea

Sphaeropsis sapinea is a fungal endophyte of Pinus spp. that can cause disease following predisposition of trees by biotic or abiotic stresses. Four morphotypes of S. sapinea have been described from within the natural range of the fungus, while only one morphotype has been identified on exotic pines in the Southern Hemisphere. The aim of this study was to develop robust polymorphic markers that could be used in both taxonomic and population studies. Inter-short-sequence-repeat primers containing microsatellite sequences and degenerate anchors at the 5' end were used to target microsatellite-rich areas in an S. sapinea isolate. PCR amplification using an annealing temperature of 49 degrees C resulted in profiles containing 5 to 10 bands. These bands were cloned and sequenced, and new short-sequence-repeat (SSR) primer pairs were designed that flanked microsatellite-rich regions. Eleven polymorphic SSR markers were tested on 40 isolates of S. sapinea representing different morphotypes as well as on 2 isolates of the closely related species Botryosphaeria obtusa. The putative I morphotype was found to be identical to B. obtusa. Otherwise, the markers clearly distinguished the remaining three morphotypes and, furthermore, showed that the C morphotype was more closely related to the A than the B morphotype. The B morphotype was the most genetically diverse, and the isolates could be further divided based on their geographic origins. Sequencing of different alleles from each locus showed that the most polymorphic markers had mutations within a microsatellite sequence.     

Biological and physiological characteristics of Neotyphodium gansuense symbiotic with Achnatherum inebrians

Biological and physiological characteristics of Neotyphodium gansuense were compared with Neotyphodium coenophialum and Epichlo? festucae at a range of temperatures and pH values, and on carbon and nitrogen amended media. N. gansuense was able to grow at 10-30 degrees C, but not at 5 degrees C, and slowly at 35 degrees C. The optimal temperature for both N. gansuense and N. coenophialum was 25 degrees C, but that of E. festucae was 20-25 degrees C. The optimal pH ranges for mycelial growth of N. gansuense, N. coenophialum and E. festucae were 5-9, 5-9 and 5-7, respectively. The Neotyphodium and Epichlo? endophytes varied in their ability to grow on media containing different carbon and nitrogen nutrients. The preference of N. gansuense for carbon source was sucrose>glucose, lactose, sorbitol, inulin, maltose, mannitol, starch, fructose>xylose. Growth of all three endophytes tested was significantly improved by peptone, tryptone, casein, yeast extract and l-proline. Yeast extract, peptone, casein, tryptone, l-proline, potassium nitrate, ammonium oxalic acid and l-leucine significantly improved growth of N. gansuense. However, ammonium nitrite was not utilized at all by any tested endophyte. N. gansuense grew significantly better on potato dextrose agar (PDA) and oat meal agar (OMA) than on corn meal agar (CMA) and drunken-horse-grass agar (DA), and most slowly on water agar (WA) and saltwater nutrient agar (SNA).     

Development and characterization of EST-derived simple sequence repeat (SSR) markers for pasture grass endophytes.

Fungal endophytes of the genus Neotyphodium are common in temperate pasture grass species and confer both beneficial and deleterious agronomic characteristics to their hosts. The aim of this study was to develop molecular markers based on simple sequence repeat (SSR) loci for the identification and assessment of genetic diversity among Neotyphodium endophytes in grasses. Expressed sequence tags (ESTs) from both Neptyphodium coenophialum and Neotyphodium lolii were examined, and unique SSR loci were identified in 9.7% of the N. coenophialum sequences and 6.3% of the N. lolii sequences. A variety of SSRs were present, although perfect trinucleotide repeat arrays were the most common. Primers were designed to 50 SSR loci from N. coenophialum and 57 SSR loci from N. lolii and were evaluated using 20 Neotyphodium and Epichlo? isolates. A high proportion of the N. coenophialum and N. lolii primers produced amplification products from the majority of isolates and most of these primers detected genetic variation. SSR markers from both N. coenophialum and N. lolii detected high levels of polymorphism between Neotyphodium and Epichlo? species, and low levels of polymorphism within N. coenophialum and N. lolii. SSR markers may be used in appropriate combinations to discriminate between species. Comparison with amplified fragment length polymorphism (AFLP) data demonstrated that the SSR markers were informative for the assessment of genetic variation within and between endophyte species. These markers may be used to identify endophyte taxa and to evaluate intraspecific population diversity, which may be correlated with variation for endophyte-derived agronomic traits.     

内生真菌感染对3个不同地理种群羽茅光合特性的影响

研究了内蒙古中东部草原3个不同样地（羊草、西乌旗和霍林河）中Neotyphodium内生真菌的侵染对宿主羽茅(Achnatherum sibiricum(L.) Keng)种群光合生理特性的影响,结果表明：自然条件下,羊草样地和西乌旗样地染菌(E+)羽茅植株的净光合速率(P_n)显著高于不染菌(E-)羽茅,说明E+羽茅植株具有更高的同化能力,而霍林河样地中二者差异不显著;3个样地中Neotyphodium内生真菌对宿主羽茅的蒸腾速率、气孔导度、光能利用效率、水分利用效率均无显著影响;引起羽茅植株午间P_n降低的主要原因是非气孔因素,与是否染菌没有必然联系;光响应曲线拟合结果表明Neotyphodium内生真菌对宿主羽茅的表观量子效率、最大净光合速率、暗呼吸速率、光补偿点和光饱和点没有显著影响。     

Simple Sequence Repeat Markers Distinguish among Morphotypes of Sphaeropsis sapinea

Sphaeropsis sapinea is a fungal endophyte of Pinus spp. that can cause disease following predisposition of trees by biotic or abiotic stresses. Four morphotypes of S. sapinea have been described from within the natural range of the fungus, while only one morphotype has been identified on exotic pines in the Southern Hemisphere. The aim of this study was to develop robust polymorphic markers that could be used in both taxonomic and population studies. Inter-short-sequence-repeat primers containing microsatellite sequences and degenerate anchors at the 5′ end were used to target microsatellite-rich areas in an S. sapinea isolate. PCR amplification using an annealing temperature of 49°C resulted in profiles containing 5 to 10 bands. These bands were cloned and sequenced, and new short-sequence-repeat (SSR) primer pairs were designed that flanked microsatellite-rich regions. Eleven polymorphic SSR markers were tested on 40 isolates of S. sapinea representing different morphotypes as well as on 2 isolates of the closely related species Botryosphaeria obtusa. The putative I morphotype was found to be identical to B. obtusa. Otherwise, the markers clearly distinguished the remaining three morphotypes and, furthermore, showed that the C morphotype was more closely related to the A than the B morphotype. The B morphotype was the most genetically diverse, and the isolates could be further divided based on their geographic origins. Sequencing of different alleles from each locus showed that the most polymorphic markers had mutations within a microsatellite sequence.     

Origin, divergence, and phylogeny of epichloe endophytes of native Argentine grasses

The epichlo? endophytes are systemic, constitutive, and often vertically transmitted fungal symbionts of grass species in subfamily Po?ideae. Prior studies indicate that several asexual epichlo? endophytes (Neotyphodium species) have evolved directly from sexual (Epichlo?) species, whereas others evolved by hybridization between two or more endophyte species. In this paper, we investigate the phylogenies of 27 Neotyphodium spp. isolates from 10 native grass species (in 4 tribes) in 22 populations throughout Argentina. Relationships among these fungi and a worldwide collection of epichlo? endophytes were estimated by phylogenetic analysis of sequences from variable portions (mainly introns) of genes for beta-tubulin (tub2) and translation elongation factor 1-alpha (tef1). Most of the Argentine endophyte isolates were interspecific hybrids of Epichlo? festucae and E. typhina. Only one isolate was a hybrid of a different ancestry, and three isolates were apparently non-hybrid endophytes. These results indicate that interspecific hybridization, which promotes genetic variation, was common during the evolution of the endophytes of Argentine grasses.     

Polymorphism in a common Atlantic reef coral (Montastraea cavernosa) and its long-term evolutionary implications

Recent advances in morphometrics and genetics have led to the discovery of numerous cryptic species in coral reef ecosystems. A prime example is the Montastraea annularis scleractinian coral species complex, in which morphological, genetic, and reproductive data concur on species boundaries, allowing evaluation of long-term patterns of speciation and evolutionary innovation. Here we test for cryptic species in the Atlantic species, Montastraea cavernosa, long recognized as polymorphic. Our modern samples consist of 94 colonies collected at four locations (Belize, Panamá, Puerto Rico in the Caribbean; S?o Tomé in the Eastern Atlantic). Our fossil samples consist of 78 colonies from the Plio-Pleistocene of Costa Rica and Panamá. Landmark morphometric data were collected on thin sections of 46 modern and 78 fossil colonies. Mahalanobis distances between colonies were calculated using Bookstein coordinates, revealing two modern and four fossil morphotypes. The remaining 48 of the 94 modern colonies were assigned to morphotype using discriminant analysis of calical measurements. Cross-tabulation and multiple comparisons tests show no significant morphological differences among geographic locations or water depths. Patterns of variation within and among fossil morphotypes are similar to modern morphotypes. DNA sequence data were collected for two polymorphic nuclear loci (β-tub1 and β-tub2) on all 94 modern colonies. Haplotype networks show that both genes consist of two clades, but morphotypes are not associated with genetic clades. Genotype frequencies and two-locus genotype assignments indicate genetic exchange across clades, and ϕst values show no genetic differentiation between morphotypes at different locations. Taken together, our morphological and genetic results do not provide evidence for cryptic species in M. cavernosa, but indicate instead that this species has an unusually high degree of polymorphism over a wide geographic area and persisting for >25 million years (myr).     

Electrophoretic variability within the Protogonyaulax tamarensis/catenella species complex: Pyridine linked dehydrogenases

Extracts from 20 isolates of the Protogonyaulax (= Gonyaulax) tamarensis/catenella species complex from diverse geographical locations, including ten contemporaneous isolates from the same geographical population, were subjected to enzyme electrophoresis. Analysis of isozyme banding patterns produced by eight pyridine-linked dehydrogenases revealed a high degree of genetic polymorphism within and between morphotypes and geographical populations. Since there was little apparent correlation between electromorphs and variants assigned provisionally to the catenelloid or tamarensoid morphotype, and because morphological intermediates exist, the presently used morphological characters used to discriminate unequivocally between P. catenella and P. tamarensis appear to be inadequate. Nevertheless, within a given morphotype, isolates from the same location were more similar than to those from elsewhere. Isozyme electrophoresis offers a means of biochemically discriminating between isolates of this species complex, for which the plate patterns are substantially the same.