Development,persistence and regeneration of foraging ectomycorrhizal mycelial systems in soil microcosms

Development of extraradical mycelia of two strains each of Paxillus involutus and Suillus bovinus in ectomycorrhizal association with Pinus sylvestris seedlings was studied in two dimensions in non-sterile soil microcosms. There were significant inter- and intra-specific differences in extraradical mycelial growth and morphology. The mycelial systems of both strains of P. involutus were diffuse and extended more rapidly than those of S. bovinus. Depending on the strain, P. involutus mycelia were either highly plane filled, with high mass fractal dimension (a measure of space filling) or sparse, low mass fractal dimension systems. Older mycelial systems persisted as linear cords interlinking ectomycorrhizal tips. S. bovinus produced either a mycelium with a mixture of mycelial cords and diffuse fans that rapidly filled explorable area, or a predominately corded mycelium of minimal area cover. In the soil microcosms, mass fractal dimension and mycelial cover tended to increase with time, mycelia encountering litter having significantly greater values. Results are discussed in terms of the ecology of these fungi, their foraging activities and functional importance in forest ecosystems.     

Lipid profile of Pleurotus sajor caju

The lipid profile of Pleurotus sajor caju was studied in relation to mycelial and sporophore growth and different cultural factors. The growth was characterised by lipid synthesis during mycelial growth and utilisation during sporophore growth. The degree of instauration increased during mycelial growth and decreased during sporophore formation. The fatty acid composition of mycelium and sporophore was similar, linoleic acid (C_18:2) being the most dominant acid in both. C:N ratio had a significant (P<0.05) positive effect on mycelial dry weight; however, per cent total lipids was similar. Non-polar lipids became more unsaturated as the temperature was raised from 10° to 25°C and pH from 3.0 to 6.0, but declined when the cultures were aerated. Mycelial dry weight increased significantly (P<0.05) when the liquid medium was supplemented with lipids. In general, fatty acids with carbon chain length C₁₆ and C₁₈ stimulated the growth of mycelium. Supplementation of solid substrate (cotton seed hulls) with safflower oil, soybean oil or rice bran significantly (P<0.05) increased the yield of sporophores. Total lipids and ratio of non-polar to polar lipids were not affected by lipid supplementation.     

甘蔗鞭黑粉菌侵染甘蔗的早期可视化观察

【目的】甘蔗鞭黑粉菌(Sporisorium scitamineum)引起的甘蔗黑穗病是我国甘蔗生产重要的病害。示踪甘蔗鞭黑粉菌侵染甘蔗的过程将有助于揭示其致病性和甘蔗抗黑穗病机制,为抗病品种的选育以及黑穗病的防治奠定基础。【方法】利用农杆菌介导的遗传转化技术对甘蔗鞭黑粉菌进行黄色荧光标记,对转化子进行配合及致病力检测,将标记菌株接种甘蔗感病品种ROC22及抗病品种中蔗1号、中蔗6号和中蔗9号并进行早期可视化观察。【结果】组成型表达的eYFP不影响标记菌株的配合及致病能力,而且黄色荧光性状能通过冬孢子稳定遗传。激光共聚焦显微观察表明,注射接种病原菌第5天,在感病品种ROC22的生长点已可见荧光菌丝及少量聚集状菌丝体,在抗病品种中蔗1号、中蔗6号和中蔗9号中可见少量单一丝状菌丝,无聚集状菌丝体。接种后35 d,在ROC22中可见大量聚集状菌丝体,但在中蔗品种中的聚集状菌丝体明显较少,而以中蔗1号最少。【结论】成功构建了甘蔗鞭黑粉菌侵染甘蔗的荧光示踪系统,并发现中蔗系列品种存在抑制甘蔗鞭黑粉菌菌丝体在细胞间扩展的机制。     

Bipolar incompatibility system of an ectomycorrhizal basidiomycete, Rhizopogon rubescens

The mating systems of most ectomycorrhizal fungi have not been elucidated because of two reasons. One is the difficulty of obtaining homokaryotic isolates for mating tests caused by the low germination rate of basidiospores, and another is the difficulty of checking dikaryotization caused by the absence or inconsistent production of clamp connections on heterokaryotic mycelia under laboratory conditions. Basidiospore germination of a few ectomycorrhizal fungi has been induced by living roots of their host plants. Based on this information, we examined methods to obtain homokaryotic isolates of Rhizopogon rubescens using its host plant, Pinus thunbergii. The basidiospores of R. rubescens appeared to germinate well on an agar plate, on which axenic pine seedlings were grown in advance to induce germination, even when the seedlings were removed from the plate at the time of spore inoculation. To enhance the production rate of clamp connections on the heterokaryotic mycelia of R. rubescens, the culture medium composition was modified. The pH of the medium was critical for the production of clamp connections, and the optimal pH was higher for the production of clamp connections than for mycelial growth. These findings made it possible to conduct mating tests, and we found that the mating system of R. rubescens is bipolar with a multiallelic mating type factor.     

High‐throughput genomics in sorghum: from whole‐genome resequencing to a SNP screening array

With its small, diploid and completely sequenced genome, sorghum (Sorghum bicolor L. Moench) is highly amenable to genomics‐based breeding approaches. Here, we describe the development and testing of a robust single‐nucleotide polymorphism (SNP) array platform that enables polymorphism screening for genome‐wide and trait‐linked polymorphisms in genetically diverse S. bicolor populations. Whole‐genome sequences with 6× to 12× coverage from five genetically diverse S. bicolor genotypes, including three sweet sorghums and two grain sorghums, were aligned to the sorghum reference genome. From over 1 million high‐quality SNPs, we selected 2124 Infinium Type II SNPs that were informative in all six source genomes, gave an optimal Assay Design Tool (ADT) score, had allele frequencies of 50% in the six genotypes and were evenly spaced throughout the S. bicolor genome. Furthermore, by phenotype‐based pool sequencing, we selected an additional 876 SNPs with a phenotypic association to early‐stage chilling tolerance, a key trait for European sorghum breeding. The 3000 attempted bead types were used to populate half of a dual‐species Illumina iSelect SNP array. The array was tested using 564 Sorghum spp. genotypes, including offspring from four unrelated recombinant inbred line (RIL) and F₂ populations and a genetic diversity collection. A high call rate of over 80% enabled validation of 2620 robust and polymorphic sorghum SNPs, underlining the efficiency of the array development scheme for whole‐genome SNP selection and screening, with diverse applications including genetic mapping, genome‐wide association studies and genomic selection.     

Element profiles and growth in Zn-sensitive and Zn-resistant Suilloid fungi

Zn pollution has triggered evolution for adaptive Zn tolerance in populations of Suilloid ectomycorrhizal fungi. The objectives of this study were to determine differential physiological responses that are linked to the Zn tolerance trait and to obtain more insight in the general mechanism responsible for the differential growth in Zn-enriched medium. Therefore, we identified intrinsic growth rates and element profiles in Zn-sensitive and Zn-tolerant genotypes. Isolates from Zn-polluted and unpolluted sites were exposed in vitro to increasing Zn²⁺ stress. The Zn concentration which inhibits growth by 50% (EC₅₀) was determined, and element (Zn, Fe, Mn, Cu, Mg, Ca and P) profiles in the mycelia were analysed. The intraspecific variation in growth rate and nutrient content of the in vitro grown mycelia is great and was not reduced in Zn-tolerant populations. The Zn resistance was not correlated to the intrinsic mycelial growth rate of the isolates or to the concentrations of the elements analysed, except for Zn. At low external Zn, Zn-resistant genotypes had lower Zn concentrations than sensitive isolates. At high external Zn, the differential Zn accumulation pattern between resistant and sensitive isolates became very prominent. Zn-exclusion mechanisms are most likely involved in the naturally selected adaptive Zn resistance. Other mechanisms of Zn detoxification such as sequestration of Zn on cell wall compounds or intracellular chelation and/or compartmentation are probably active but cannot explain the differential Zn sensitivity of the isolates.     

Production of enokipodins A, B, C, and D: a new group of antimicrobial metabolites from mycelial culture of Flammulina velutipes

Antimicrobial compounds enokipodins A, B, C, and D were originally isolated from the culture filtrates of Flammulina velutipes mycelial culture. Analysis of antibacterial activity by the paper disk method and quantification of enokipodins A–D by high performance liquid chromatography (HPLC) showed that F. velutipes mycelia produced enokipodins A–D in their late growing phase. Great genetic variability in production of these compounds was observed among ten strains of F. velutipes in analyses of antimicrobial activity by the hole-plate diffusion method and quantification by HPLC. Enokipodins A–D demonstrated antimicrobial activity mainly against the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus. Evaluation of minimum inhibitory doses (MIDs) showed that MIDs of enokipodins A and C for B. subtilis were as low as that of the penicillin G antibiotic.     

Grazing by Folsomia candida (Collembola) differentially affects mycelial morphology of the cord-forming basidiomycetes Hypholoma fasciculare, Phanerochaete velutina and Resinicium bicolor

Cord-forming basidiomycetes are important decomposers of dead wood in forest ecosystems but the impact of mycophagous soil invertebrates on their mycelia are little known. Here we investigate the effects of different grazing intensities of Collembola (Folsomia candida) on mycelial foraging patterns of the saprotrophic cord-forming basidiomycetes Hypholoma fasciculare, Phanerochaete velutina and Resinicium bicolor growing from beech (Fagus sylvatica) wood block inocula in dishes of non-sterile soil. Mycelial extension rate and hyphal coverage decreased with increased grazing intensity. R. bicolor was most affected, high grazing density resulting in only a few major cords remaining. Grazing of H. fasciculare often resulted in points of more rapid outgrowth as cords with a fanned margin. In grazed mycelia of P. velutina the main cords had fanned tips and lateral cords became branched. These results suggest that mycophagy by Collembola may hinder the growth of cord-forming fungi in woodlands, which might impact on the ability of these fungi to forage for and decompose dead organic material.     

Effect of phosphate deficiency on growth and protein profile in three strains ofPholiota nameko

Changes in mycelial dry weight and soluble protein amounts and acid phosphatase activities on a mycelial dry weight basis in the mycelia and culture supernatants during the Pi-supplied (P⁺) and Pi-depleted (P⁻) cultures of three strains ofPholiota nameko were examined. Mycelial dry weights of the three strains were lower in the P⁻ culture than in the P⁺ culture. However, soluble protein amounts in the culture supernatants and acid phosphatase activities in the mycelia and culture supernatants of the three strains were higher on a mycelial dry weight basis in the P⁻ culture than in the P⁺ culture. Total proteins of strains N2 and N4 were analyzed by two-dimensional-PAGE. Comparison of electrophoretograms of the P⁺ and P⁻ cultures showed that many polypeptides in the two strains were induced and secreted by Pi deficiency, but more than half of them were specific to each strain. Activity staining of acid phosphatase also revealed that two isozymes with the same molecular weights in the three strains were induced and secreted by Pi deficiency. Adaptive mechanisms for Pi deficiency in the three strains were discussed.     

Recycling spent larval food of Corcyra cephalonica Stainton for preparing spawn and sporophore of Pleurotus sajor-caju (Fr.) Singer

Large scale production of the rice moth, Corcyra cephalonica Stainton in pearl millet grain medium leads to a huge accumulation of spent larval medium in commercial insectaries. We attempted bioconversion of spent larval medium of C. cephalonica (CLM) for cultivation of the mushroom Pleurotus sajor-caju (Fr.) Singer, to increase the usage of these residues. Maximum efficiency limits of CLM for spawn run, sporophore cropping and as bed substrate were assessed with varying combinations of sorghum and rice straw. Sorghum grains and rice straw were the best substrates for spawn run and sporophore yield respectively. Having been crushed, macerated, heated and sterilized, CLM could also become a suitable substrate along with sorghum or rice straw. Sorghum and CLM at 16.7% + 83.3% and 33.3% + 66.7% combinations were very effective in supporting mycelial growth and quicker colonization of fungus, and mother spawn yield. The spawn that was obtained from these combinations yielded higher sporophore as well. The fungus did not rapidly colonize on other combinations (50% + 50%, 66.7% + 33.3% and 83.3% + 16.7%), and was completely unable to grow on CLM 100%. Combination of rice straw and CLM at 75% + 25% and 50% + 50% as bed substrate contributed higher sporophore yield. Analysis of the substrates indicated variation in their chemical and mineral composition, but they were good sources of N, P and Ca. The prospects of exploring CLM for the mushroom cultivation are discussed.     

Genetic variability of phosphate solubilizing activity by monocaryotic and dicaryotic mycelia of the ectomycorrhizal fungus Laccaria bicolor (Maire) P.D. Orton

Homocaryotic mycelia obtained from spores of Laccaria bicolor S238 have been compared in vitro for their efficiency in solubilizing poorly soluble phosphates. This could lead to genetic selection according to such criteria. However, there is very little room for improvement as the wild strain was shown to be one of the most efficient solubilizers among the strains tested. Twenty dicaryotic strains obtained by crossing the compatible homocaryons have also been compared and no clear heritability of this character has been found. The four phosphate salts used are most probably solubilized by the same mechanism which is polygenetically controlled     

Comparisons of methods for introgressing exotic germ plasm into adapted sorghum

The incorporation of exotic germ plasm into breeding populations can broaden and diversify the genetic base of adapted genotypes. To more effectively utilize the genetic resources existing in Sorghum bicolor (L.) Moench, a rapid and efficient method of incorporating exotic genotypes into adapted populations is needed. Therefore, this study was conducted to compare the effectiveness of backcrossing to a broad-based population versus backcrossing to an inbred line for developing improved lines from adapted x exotic crosses. A wild sorghum, a cultivated landrace, and a converted sorghum line were crossed to an inbred line (CK60) and a broad-based population (KP9B). After two generations of backcrossing to the respective adapted parent, 50 F₂ lines were derived from each of the backcross generations of every mating and evaluated at three test environments. Backcrossing to an inbred line (CK60) gave fewer high-yielding segregates and generated less genetic variation than backcrossing to a population (KP9B). Also, the number of agronomically acceptable lines derived from each CK60 mating was fewer than that derived from the corresponding mating with KP9B. Overall, the use of a broad-based population as an adapted recurrent parent for introgressing exotic genotypes may provide good opportunities for developing suitable inbred lines from adapted x exotic backcrosses.Contribution no. 93-499-J from the Kansas Agricultural Experiment Station     

Genetic and clonal diversity in Korean populations ofVitex rotundifolia (Verbenaceae)

Vitex rotundifolia L.f. is a woody perennial and has sexual and asexual modes of reproduction. Allozyme study was conducted on 550 plants in 13 Korean populations. The levels of genetic variability and divergence within and among populations, respectively, are considerably lower and higher than the mean values for woody plants with similar life history tralts. Mean percentage of polymorphic loci (P _P), mean number of alleles per locus (A _P), and mean genetic diversity (He _P) within populations ofV. rotundifolia were: 16.7%, 1.21, and 0.047. On average, about 79% of the total variation inV. rotundifolia was common to all populations (meanG _ST=0.208). In addition, significant differences in allele frequencies among populations were found in all polymorphic loci examined (P<0.001). On the other hand, levels of genotypic diversity within and among populations were moderate. About 44% (18/41) of multilocus genotypes were “local genotypes” (genotypes occurring in only one population), whereas only one “widespread genotype” (genotypes occurring in more than 75% of the populations) were detected. The mean number of multilocus genotypes per population (G) and mean genotypic diversity index (D _G) were 8.4 and 0.74, respectively. Most common multilocus genotypes found in populations were homozygous for five polymorphic loci. The abundance of ramets of these genets is responsible for the low levels of expected heterozygosity within populations. The results indicate that clonal reproduction may act as an enhancer of genetic drift by reducing effective size of local populations ofV. rotundifolia.     

Evidence of concurrent local adaptation and high phenotypic plasticity in a polar microeukaryote

Here we investigated whether there is evidence of local adaptation in strains of an ancestrally marine dinoflagellate to the lacustrine environment they now inhabit (optimal genotypes) and/or if they have evolved phenotypic plasticity (a range of phenotypes). Eleven strains of Polarella glacialis were isolated and cultured from three different environments: the polar seas, a hyposaline and a hypersaline Antarctic lake. Local adaptation was tested by comparing growth rates of lacustrine and marine strains at their own and reciprocal site conditions. To determine phenotypic plasticity, we measured the reaction norm for salinity. We found evidence of both, limited local adaptation and higher phenotypic plasticity in lacustrine strains when compared with marine ancestors. At extreme high salinities, local lake strains outperformed other strains, and at extreme low salinities, strains from the hyposaline lake outperformed all other strains. The data suggest that lake populations may have evolved higher phenotypic plasticity in the lake habitats compared with the sea, presumably due to the high temporal variability in salinity in the lacustrine systems. Moreover, the interval of salinity tolerance differed between strains from the hyposaline and hypersaline lakes, indicating local adaptation promoted by different salinity.     

Genetic analysis of morphological variability in three Plantago species with different mating systems

Summary Morphological variability in three Plantago species possessing different mating systems — P. Major, P. coronopus, and P. lanceolata — was analyzed. From each species several populations were analyzed. In comparing the species, it appeared that both the inbreeding P. major and the outbreeding P. lanceolata showed a high degree of morphological differentiation between populations. It was hypothesized that the absence of population differentiation for the species with a mixed mating system, P. coronopus, was caused by high adaptive plasticity. Plasticity is probably the predominant mechanism by which this species copes with different environments. There were considerable differences between populations within each species for their levels of variability and their heritabilities. It appeared that besides the influence of the mating system, selection may diminish morphological variability in the case of strong directional selection, especially in P. major and P. lanceolata.Grassland Species Research Group Publication No. 135     

Assessment ofGibberella fujikuroi mating type by PCR

Mating type (MAT)-specific fragments of the two idiomorphs ofGibberella fujikuroi (anamorph,Fusarium moniliforme) were obtained by PCR amplification using primers to conserved regions ofMAT homologs from other fungal species and used to assign mating type by molecular criteria rather than the arbitrary historical designation. Mating type—strains of mating populations A-E and a mating type+strain of mating population F carry an α-box motif and should therefore be designatedMAT-1. Mating type+strains of mating populations A-E and a mating type—strain of mating population F carry an HMG-box motif and should be designatedMAT-2. Thus, assessment of mating type ofG. fujikurol strains can be easily achieved usingMAT-specific primers.     

Improving dispersed growth of Frankia using Carbopol

In laboratory culture, strains of the actinomycete Frankia grow slowly and form dense mycelial pellets. In this study, we have shown that by adding the anionic polymer Carbopol 941® to liquid growth media, dispersed growth of Frankia is improved. Cell protein measurements indicated higher biomass production in cultures maintained in media supplemented with Carbopol. Fluorescence staining and microscopy were used to compare viability of well-dispersed mycelia and pellets.     

荧光蛋白在双色蜡蘑中的构建与表达

菌根是真菌与植物之间形成的互惠互利的营养共生体,对生态环境有重大的意义。外生菌根真菌与植物互作机制以及真菌基因功能的深入研究都需要对菌根真菌进行遗传转化,本研究以外生菌根真菌模式生物双色蜡蘑(Laccaria bicolor)为研究对象,选择细胞核中的核小体蛋白H₂B基因为目的基因,以pCEBN为表达载体,融合红色荧光蛋白,最终构建在真菌中表达的双元载体,使用根瘤农杆菌介导转化法转化双色蜡蘑菌丝,随后利用PCR对真菌转化子进行验证后,通过激光共聚焦显微镜观察到菌丝细胞核中的红色荧光,成功将融合荧光蛋白转化菌根真菌,为后续研究菌根真菌中基因的亚细胞定位提供了实验平台。结果表明,利用双元载体和农杆菌转化方法,建立了高效的双色蜡蘑转化体系(93.33%),在激光共聚焦显微镜下观察到菌丝细胞核中红色荧光信号,验证了融合荧光蛋白在双色蜡蘑中的成功表达。本研究成功地构建了菌根真菌中的核小体蛋白和红色荧光蛋白融合表达的真菌转化体系。     

Allozyme variation in the sympatric ferns Culcita macrocarpa and Woodwardia radicans at the northern extreme of their ranges

Isozymes were used to study genetic variation in the clonal ferns Culcita macrocarpa and Woodwardia radicans in the northwestern Iberian Peninsula, their northern distributional limit. Despite their high chromosome numbers, both species were isozymic diploids. In C. macrocarpa all 18 resolved loci were monomorphic, with the same allele in all ramets from all populations. In W. radicans only two of the 16 interpreted loci were polymorphic, with two alleles per locus; ramet-level genotypes showed Hardy-Weinberg equilibrium, indicating an intergametophytic mating system; the number of genets distinguished was 1–3 per population; and among-population variation was low (F _ST = 0.231), suggesting effective gene flow (i.e. spore exchange). More generally, the very low (W. radicans, H _T = 0.012) or zero (C. macrocarpa) genetic diversity detected in the present study may be due to genetic drift associated with the reduction of populations in the last glaciation, and to founder effects in the subsequent Holocene expansion.     

Frequent diploidisation of haploid Armillaria ostoyae strains in an outdoor inoculation experiment

Very little is known about the biology and ecology of haploid Armillaria strains in nature. In this outdoor inoculation experiment, we assessed the virulence of six haploid Armillariaostoyae strains along with their diploid parent towards 2-year-old seedlings and 4-year-old saplings of Norway spruce (Picea abies), and determined their ability to colonise freshly cut stumps. As inoculum source an Armillaria-colonised hazelnut (Corylus avellana) stem segment was inserted into the soil substrate. Re-isolations from mycelial fans at the root collar of infected trees or stumps were made. Surprisingly, not a single haploid re-isolate could be recovered. Microsatellite genotyping of 133 re-isolates suggests that the inoculated haploid strains were diploidised either by mating propagules (basidiospores or haploid mycelia) already present in the soil substrate or naturally disseminated in the course of the experiment from nearby forests. Consequently, no conclusion about the infectious ability of haploid Armillaria mycelia under natural conditions can be drawn. Nonetheless, the diploid half-sib families resulting from the diploidisation showed varying degrees of virulence, with a high correlation between the experiment with 2-year-old seedlings and 4-year-old saplings. Despite extensive genotyping of re-isolates, no evidence for somatic recombination between haploid mating propagules and diploidised mycelia was detected, suggesting that this is an uncommon phenomenon in A. ostoyae.