Systematics of AustralianLepidium species (Brassicaceae) and implications for their origin: Evidence from IEF analysis of Rubisco

Variation in subunit polypeptide composition of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from native AustralianLepidium taxa was analyzed by isoelectric focusing (IEF). Species of grexMonoplocoidea on the one hand, and species of gregesPseudoruderalia andPapillosa on the other, were well separated by Rubisco IEF patterns. The protein data suggest grexMonoplocoidea to be placed in sect.Monoploca whereas gregesPseudoruderalia andPapillosa should be integrated into sect.Dileptium. We suggest that the Rubisco type of American and/or AsianLepidium species was contained in those plants migrating to Australia and evolving into the recent native Australian species. Fossil pollen records and vegetation history indicate that the origin ofLepidium in Australia was restricted to the late Tertiary of Quaternary. Data presented in this study fit best with an immigration by long-distance dispersal of seeds by birds from Asia and/or from South America.     

Similar experiment – different conclusion; a response to the recent proposition of Septoria lepidii as a potentially important biological control agent for weedy species of Lepidium in North America

Following preliminary host-specificity testing Septori lepidii Desm. was recently proposed as a potentially important biological control agent for invasive Lepidium species in North America. Conversely, a previous, unpublished assessment of S. lepidii for biocontrol of Lepidium latifolium found the pathogen to be unsuitable due to infection of the American native Lepidium huberi.     

A bicontinental origin of polyploid Australian/New Zealand Lepidium species (Brassicaceae)? Evidence from genomic in situ hybridization

Background and Aims

Incongruence between chloroplast and nuclear DNA phylogenies, and single additive nucleotide positions in internal transcribed spacer (ITS) sequences of polyploid Australian/New Zealand (NZ) Lepidium species have been used to suggest a bicontinental hybrid origin. This pattern was explained by two trans-oceanic dispersals of Lepidium species from California and Africa and subsequent hybridization followed by homogenization of the ribosomal DNA sequence either to the Californian (C-clade) or to the African ITS-type (A-clade) in two different ITS-lineages of Australian/NZ Lepidium polyploids.

Methods

Genomic in situ hybridization (GISH) was used to unravel the genomic origin of polyploid Australian/NZ Lepidium species. Fluorescence in situ hybridization (FISH) with ribosomal DNA (rDNA) probes was applied to test the purported ITS evolution, and to facilitate chromosome counting in high-numbered polyploids.

Key Results

In Australian/NZ A-clade Lepidium polyploids, GISH identified African and Australian/NZ C-clade species as putative ancestral genomes. Neither the African nor the Californian genome were detected in Australian/NZ C-clade species and the Californian genome was not detected in Australian/NZ A-clade species. Five of the eight polyploid species (from 7x to 11x) displayed a diploid-like set of rDNA loci. Even the undecaploid species Lepidium muelleriferdinandi (2n = 11x = 88) showed only one pair of each rDNA repeat. In A-clade allopolyploids, in situ rDNA localization combined with GISH corroborated the presence of the African ITS-type.

Conclusions

The nuclear genomes of African and Australian/NZ C-clade species were detected by GISH in allopolyploid Australian/NZ Lepidium species of the A-clade, supporting their hybrid origin. The presumed hybrid origin of Australian/NZ C-clade taxa could not be confirmed. Hence, it is assumed that Californian ancestral taxa experienced rapid radiation in Australia/NZ into extant C-clade polyploid taxa followed by hybridization with African species. As a result, A-clade allopolyploid Lepidium species share the Californian chloroplast type and the African ITS-type with the C-clade Australian/NZ polyploid and African diploid species, respectively.Key words: Lepidium, Brassicaceae, FISH, GISH, hybridization, polyploidy, long-distance dispersal, ITS, rDNA, Australia, New Zealand     

The Growth of the Primary Roots and Root Hairs of Sinapis alba and Lepidium sativus in Triton X-100

Seeds of Lepidium and Sinapis were germinated and grown for 3 days in different concentrations of Triton X-100 (0.001–0.1 % v/v). The elongation of the primary root was slightly stimulated by low concentrations. In concentrations above 0.01 %, Triton inhibited root growth and forked root hairs developed. The hairs elongated both at the apex and at the base, exhibited protoplasmic streaming and activity of particulate non-specific esterase. In contrast the growth of the hypocotyl of both Lepidium and Sinapis diminished steadily in increasing concentrations of Triton. Triton also affected the percentage germination of Lepidium, which increased or decreased according to the concentration used. The changes in root growth and germination and the appearance of branched root hairs in abundance coincide with a change in the detergent solution from monomer to aggregated molecules.     

Host Range of Mycoleptodiscus terrestris, a Microbial Herbicide Candidate for Eurasian Watermilfoil, Myriophyllum spicatum

A native strain of the fungal plant pathogen Mycoleptodiscus terrestris is capable of causing under experimental conditions necrotic shoot lesions and a generalized decline and disintegration of Eurasian watermilfoil (Myriophyllum spicatum), an exotic submerged aquatic weed in North America. The potential of this fungus to cause disease on nontarget plants was evaluated on 33 species and cultivars in 11 families. The nontarget selection, intended as a Tier I group, was biased toward probable suscepts consisting of submerged, floating, and emergent aquatic species and terrestrial crop plants. The plants were exposed to the fungal mycelium formulated in alginate beads. Pathogenicity rather than phytotoxicity was the primary mode of attack by the fungus. Of the 16 nontarget aquatic species tested, the fungus was pathogenic to Hydrilla verticillata (hydrilla), Myriophyllum aquaticum (parrotfeather), and Ceratophyllum demersum (coontail), but only in hydrilla did it cause plant mortality comparable to that in Eurasian watermilfoil. The remaining 13 submerged, floating, and emergent aquatic species were unaffected. Of the 17 terrestrial species screened, none was significantly affected by the fungus in germination studies, but postemergent disease symptoms developed on seedlings of 10 species. Four of the 10, Medicago sativa (alfalfa), Lotus corniculatus (birdsfoot trefoil), Trifolium hybridum (alyce clover), and T. repens (white clover), developed disease affecting 26-50% of their tissues. Thus, this host range testing scheme, based on the concept of screening plants under the maximum hazard potential, helped to identify susceptible species. Nonetheless, it exaggerated the nontarget risk due to the small number of host species tested. Screening a taxonomically diverse and larger selection of plants as well as testing under conditions of less severe exposure to the fungal inoculum are necessary to obtain a more realistic view of the host range than presently indicated.     

Effect of temperature on growth and nitrogen mineralization of fungi and fungal-feeding nematodes

Dish and column microcosms containing alfalfa-sand medium were established to determine effect of temperature on growth and N mineralization ability of two fungi (Rhizoctonia solaniand Botrytis cinerea) and two nematodes (Aphelenchus avenaeand Aphelenchoides composticola). The microcosms were incubated at 15, 20, 25 and 29 °C for 21 days. In the dish microcosms, hyphal growth rates of both fungal species increased with temperature in the range of 15–25 °C. Above that temperature range, the growth rate of R. solani remained almost constant while that of B. cinereadecrease considerably. The population growth rate of A. avenae increased with temperature between 15 and 29 °C on colonies of R. solani and B. cinerea in dish microcosms. The growth rate of A. composticola also increased in the range of 15–25 °C but decreased greatly beyond that temperature range independent of the fungal species as food source. Inorganic N (NH₄ ⁺ + NO₃ ^–) was collected from each column microcosm by leaching every 3 days. In the columns containing R. solani, there was a significant effect of temperature on the amount of N detected in the fungus+A avenaeor A. composticolabut not in the fungus alone columns. The total amount of N was greatest at 29 °C for A. avenaeand at 20 °C for A. composticola columns, concurrent with the population growth rates of the nematodes. In the columns containing B. cinerea, the effect of temperature on the amount of inorganic N was not significant in either the fungus alone or fungus+nematode columns, although the population growth rates of the both nematode species were highest at 20 °C. For B. cinerea, the N amount across temperatures was the same or larger for the fungus alone as for the fungus+nematode columns. In general, the contribution of fungal-feeding nematodes to N mineralization was small in any combinations of fungus and nematode species at any temperature. Similarity in C/N ratio of the fungal and nematode biomass, organic substrate C/N ratios too low for measurable increase in net mineralization by the nematodes and small reproduction of the nematodes in the column microcosms were probable contributory factors.     

Laboratory growth rates of six species of freshwater Gymnamoebia

Summary Amaranthus and several other wind-pollinated species of plants are used to test some of the theoretical models of relative reproductive effort towards the male and female sexes. Consistent with these models, in self-compatible, monoecious Amaranthus, Chenopodium, Digitaria, Setaria, and Lepidium, female effort represented over 90% of the total reproductive effort. Also consistent with predictions, Lolium, a self-incompatible wind-pollinated species, was found to have about equal male and female effort. A method is described here that should prove useful in quantifying male and female effort in both wind and insect-pollinated species of plants.     

Distribution of Gibberellins and Abscisic Acid in Geotropically Stimulated Vicia faba Roots

The occurrence of gibberellins and abscisic acid (ABA) in extracts of roots of Vicia faba was demonstrated by gas-liquid chromatography (GLC) of the methylated eluates from the relevant zones of thin-layer chromatograms (TLC) of purified extracts. Quantitative determination of the hormone contents in extracts from upper and lower halves of roots which had been kept in the horizontal position for 30 min indicated a redistribution of the hormones during the geotropic stimulation. Gibberellins whose methyl esters appeared at the retention time of methylated gibberellic acid (GA₃), used as a standard, occurred in higher concentration in the upper than in the lower halves (ratio 2.08:1), whereas the concentration of ABA was highest in the lower halves (ratio 3.08:1). The ratio of the hormones in right and left halves of vertical roots was close to 1:1. Indoleacetic acid (IAA) and ABA were found to retard the elongation of roots of Vicia faba and Lepidium during the first 24 h. Additional experiments with Lepidium showed that this retardation occurs within the first hour after application. Low concentrations of GA₃, when applied to germinating seeds just after the radicles had broken the seed coat, stimulated root elongation in Vicia faba within 24 h and in Lepidium within 36 h. When applied to Lepidium seedlings with 20 mm long roots, GA₃ showed a stimulatory trend within the first 2 h, and distinct stimulation in the subsequent hours, particularly at the lowest concentrations, 0.01 and 0.001 mg/1. These results suggest the possibility of a participation of ABA and gibberellins (in addition to IAA) in the development of the positive geotropic curvature.     

Microsatellite primers for fungus‐growing ants

We isolated five polymorphic microsatellite loci from a library of two thousand recombinant clones of two fungus‐growing ant species, Cyphomyrmex longiscapus and Trachymyrmex cf. zeteki. Amplification and heterozygosity were tested in five species of higher attine ants using both the newly developed primers and earlier published primers that were developed for fungus‐growing ants. A total of 20 variable microsatellite loci, developed for six different species of fungus‐growing ants, are now available for studying the population genetics and colony kin‐structure of these ants.     

DNA fingerprinting of Peronospora parasitica,a biotrophic fungal pathogen of crucifers

The fungus Peronospora parasitica (Pers. ex Fr.) Fr. is an obligate biotroph infecting a wide range of host species in the family Cruciferae. Isolates from different hosts are morphologically similar, and pathotypes are usually distinguished on the basis of host range. Random Amplified Polymorphic DNA (RAPD) fingerprints were generated from a range of P. parasitica isolates from different Brassica species. Reaction conditions, in particular DNA template, primer and Mg²⁺ concentrations, were optimized to ensure that amplifications were reproducible. Possible artefacts arising through host plant DNA were assessed by including such DNA in control reactions. Confirmation that diagnostic RAPD bands were generated from fungal DNA was also obtained by Southern hybridization of a RAPD band to genomic fungal DNA. By screening 20 decamer primers, 2 were found to detect sufficient genetic variation to allow complete differentiation between pathotypes. These results illustrate the potential value of RAPDs for detecting polymorphisms between isolates of a non-culturable plant pathogenic fungus.     

Seasonal dynamics of non-structural carbohydrates in two species of mediterranean sub-shrubs with different leaf phenology

The seasonal dynamics of non-structural carbohydrates in the woody organs of two co-existing mediterranean sub-shrubs were analyzed. The two species show different leaf phenology during summer: Linum suffruticosum, maintains many of its green leaves, while Lepidium subulatum sheds most of its leaves. These different leaf phenologies are related to different strategies with regard to summer stress. The maintenance of leaves in Linum is related to its stress tolerance while Lepidium avoids stress by shedding its leaves. The main objectives were to: (1) determine the differences in the seasonal dynamics of non-structural carbohydrates among the main woody organs of both species; (2) verify if differences in the leaf phenology, and hence in the strategy with regard to summer drought, lead to different seasonal patterns of carbohydrate storage and use between the two species; (3) compare the seasonal dynamics of carbohydrates of the two studied sub-shrubs with those of mediterranean trees and shrubs previously reported in the literature. The concentration of soluble sugars (SS), starch and total non-structural carbohydrates (TNC) were assessed monthly, over 17 months, in the main roots, stems and the transition zone between root and shoot systems of both species. Starch storage capacity and SS, starch and TNC pools were calculated. The seasonal pattern of carbohydrate accumulation was similar among the woody organs analyzed, but it differed with those reported for mediterranean trees and shrubs. The two species showed different pools and seasonal patterns of non-structural carbohydrate concentrations in its woody organ, which corresponded to their different extent of leaf shedding. The stress-avoider Lepidium accumulated starch during spring shoot growth as a carbon store for summer respiration and had low pools of SS, whereas the stress-tolerant Linum increased SS during summer drought to maintain photosynthetic activity during summer and had low starch pools and storage capacity. However, irrespective of their different leaf shedding patterns, both species had a similar relative variation of their TNC concentration, which contrasts with previous results on deciduous and evergreen woody species.     

Pathogenicity and Host Specificity of Mycovellosiella lantanae var. lantanae,a Potential Biocontrol Agent for Lantana camara in South Africa

Lantana camara is a poisonous, bushy shrub from South and Central America that has invaded much of the moist, warm sub-tropical areas of South Africa. In the last decade, evidence of a conspicuous and damaging mycobiota on lantana in the Neotropics has persuaded biocontrol researchers to consider fungi as potential biocontrol agents for this plant. Samples of diseased L. camara leaves were collected during field surveys in South, North and Central America from 1987 to 1997. Pathogenicity tests showed the leaf spot fungus, Mycovellosiella lantanae var. lantanae to be a promising biocontrol pathogen. Inoculations onto South African biotypes of L. camara and a number of closely related species within the family Verbenaceae were made under quarantine conditions to determine the pathogenicity and host specificity of the fungus. Inoculation of L. camara biotypes resulted in necrotic, grey lesions (20-60 per leaf), necrosis of flower buds and stalks, as well as defoliation of certain biotypes after three weeks. None of the other plant species tested showed any disease symptoms. This indicates a very restricted host range, making this pathogen a suitable candidate for application as a biological control agent. It is expected that the fungus will reduce the vigour and reproductive potential of L. camara. Due to the variation in virulence of M. lantanae var. lantanae when tested on South African lantana biotypes, release strategies may include the use of combinations of the isolates to target a wider range of lantana biotypes in the field. Permission to release M. lantanae var. lantanae in South Africa was granted in September 2001. The pathogen will be released as a classical biocontrol agent in South Africa.     

Host specificity of <Emphasis Type="Italic">Euops chinesis</Emphasis>, a potential biological control agent of <Emphasis Type="Italic">Fallopia japonica</Emphasis>, an invasive plant in Europe and North America

Fallopia japonica (Houttuyn) Ronse Decraene (Polygonaceae) is a serious invasive weed in North American and Europe. In its native China, a leaf-rolling weevil, Euops chinesis (Coleoptera: Attelabidae) was found attacking F. japonica in the field. No-choice tests, multiple-choice tests, open field tests and field surveys were conducted as a measure of its host specificity. Forty-six plant species were selected from 17 families for host range testing, among which, six species, F. multiflora, F. japonica, Persicaria perfoliata, Rumex acetosa, R. japonicus and R. aquaticus, were exposed to adults in no-choice tests. However, larvae could only develop successfully on F. japonica, and this plant appeared to be the only host in the field, suggesting the weevil is host-specific. As larval development appears to depend on a fungus in the leaf rolls, the insect–fungus mutualism and risks including host specificity of the fungus should be evaluated before the insect’s introduction.     

Puccinia kraussiana (Uredinales) onHeterosmilax gaudichaudiana,A new record of rust in Eastern Asia

Puccinia kraussiana, a rust fungus of Africa, was found in Taiwan for the first time in eastern Asia. It was parasiting on leaves ofHeterosmilax gaudichaudiana, on which all four stages were characteristically found. The host species,H. gaudichaudiana, newly added to the host range ofP. kraussiana, constitutes also a new entry to the phanerogamic flora of Taiwan.     

Fungus Symbionts Colonizing the Galleries of the Ambrosia Beetle <Emphasis Type="Italic">Platypus quercivorus</Emphasis>

Isolations were made to determine the fungal symbionts colonizing Platypus quercivorus beetle galleries of dead or dying Quercus laurifolia, Castanopsis cuspidata, Quercus serrata, Quercus crispula, and Quercus robur. For these studies, logs from oak wilt-killed trees were collected from Kyoto Prefecture, Japan. Fungi were isolated from the: (1) entrances of beetle galleries, (2) vertical galleries, (3) lateral galleries, and (4) the larval cradle of P. quercivorus in each host tree. Among the fungus colonies which appeared on YM agar plates, 1,219 were isolated as the representative isolates for fungus species inhabiting in the galleries based on their cultural characteristics. The validity of the visual classification of the fungus colonies was checked and if necessary properly corrected using microsatellite-primed PCR fingerprints. The nucleotide sequence of the D1/D2 region of the large subunit nuclear rRNA gene detected 38 fungus species (104 strains) of which three species, i.e., Candida sp. 3, Candida kashinagacola (both yeasts), and the filamentous fungus Raffaelea quercivora were isolated from all the tree species. The two yeasts were most prevalent in the interior of galleries, regardless of host tree species, suggesting their close association with the beetle. A culture-independent method, terminal restriction fragment length polymorphism (T-RFLP) analysis was also used to characterize the fungus flora of beetle galleries. T-RFLP patterns showed that yeast species belonging to the genus Ambrosiozyma frequently occurred on the gallery walls along with the two Candida species. Ours is the first report showing the specific fungi inhabiting the galleries of a platypodid ambrosia beetle.     

Isozyme evidence for host races of the fungus Atkinsonella hypoxylon (Clavicipitaceae) infecting the Danthonia (Poaceae) complex in the southern Appalachians

On granite outcrops around Highlands, North Carolina four sympatric Danthonia grass species are infected by the fungus Atkinsonella hypoxylon (Clavicipitaceae). Danthonia epilis and D. sericea (rare hosts) are infected only in this small region, although they range more widely, while D. compressa and D. spicata (widespread hosts) are infected throughout their ranges in eastern North America. To test the hypothesis that infection of the rare hosts has recently spread from the widespread hosts, the genetic structure of fungal populations on different Danthonia hosts was investigated by isozyme electrophoresis. Two hundred isolates were collected from 46 sites and analyzed for ten polymorphic isozyme loci to examine levels of variation, genetic differentiation, and gene flow among fungal populations infecting different hosts. Genetic identities (I) indicated that isolates from within each of the widespread and rare host species pairs were homogeneous (I > 0.99), but the pairs were highly differentiated (0.66 < I < 0.69). With one exception there was no overlap in fungal genotypes isolated from the widespread hosts (12 genotypes) vs. the rare hosts (seven genotypes). Two plants of D. compressa growing sympatrically with infected D. epilis at one site were infected with a genotype characteristic of the latter species. Estimates of gene flow suggest a high degree of host-mediated reproductive isolation in A. hypoxylon, indicating that the fungus forms distinct, long-standing host races or sibling species in the southern Appalachians.     

Isolation of 21 new polymorphic microsatellite loci in the phytopathogenic fungus Venturia inaequalis

Twenty‐one new polymorphic microsatellite markers were isolated in the phytopathogenic fungus Venturia inaequalis, the causal agent of apple scab. An enrichment protocol was used to isolate microsatellite loci and the level of polymorphism was assessed on 44 European isolates. All loci were polymorphic with an average of 9.1 alleles per locus (range 2–24). Tests of cross‐species amplifications suggest that at least some of these microsatellites could be used in different species, mainly Spilocaea pyracanthae and S. eriobotryae.     

中国独行菜属种皮微形态特征的研究

利用扫描电子显微镜对独行菜属9种植物种子的种皮微形态进行了研究,结果表明独行菜属种皮微形态可划分为4种类型;结果支持将独行菜属划分为3个组的观点;北美独行菜在种皮纹饰上与其它类群差别较大,并结合子叶缘倚胚根的特征将其另立一新组——子叶缘倚组Sect. Accumbentum F. Z. Li, J. Q. Zheng ＆ Z. Y. Sun , sect. nov.     

Genetic diversity of Ophiocordyceps sinensis, a medicinal fungus endemic to the Tibetan Plateau: Implications for its evolution and conservation

Background  

Ophiocordyceps sinensis (syn. Cordyceps sinensis), endemic to alpine regions on the Tibetan plateau, is one of the most valuable medicinal fungi in the world. Huge commercial demand has led to excessive harvest and a dramatic decline in its numbers. The diversity of terrains and climates on the Tibetan Plateau and the broad insect host range (more than 50 species in the family Hepialidae) may have resulted in substantial intraspecific genetic diversity for this fungus. The objective of this study was to evaluate the population distribution of O. sinensis from geographically diverse regions of the Tibetan Plateau based on nrDNA ITS and MAT1-2-1 gene sequences. Understanding of the genetic diversity and genesis of O. sinensis will provide important information for the evolution and conservation of this fungus.