Identification of Tuber magnatum Pico DNA markers by RAPD analysis

Summary Different species of truffle were studied in order to identify species-specific markers. The isolation of two Tuber magnatum Pico markers is reported. One of these could be used as a probe in dot blot hybridization, allowing the development of a rapid test able to identify Tuber magnatum species.     

Ectomycorrhizal fungi with edible fruiting bodies 3.Tuber magnatum, tuberaceae

The Italian white truffle(Tuber magnatum Pico) forms mycorrhizal relationships, with the roots of, for example, poplars, willows, oaks, aspen, alder and hazelnut in Northern Italy and in small areas of Southern France, Switzerland and Yugoslavia. Its fruiting bodies, which are harvested in autumn and early winter, have a strong aroma and taste and are much sought-after by chefs and gourmets. Because they do not preserve well, good quality Italian white truffle is unavailable for much of the year. The vegetation, climate and soils where T. magnatum grows in Italy are described and compared with those found in similar areas in New Zealand. Market information and methods for cultivatingT. magnatum are also presented.     

Random amplified polymorphic DNA (RAPD) markers for genetic analysis in micropropagated plants of Populus deltoides Marsh

RAPD markers were used to assess genetic fidelity of 23 micropropagated plants of a single clone (L34) of Populus deltoides. Eleven arbitrary 10-base primers were successfully used to amplify DNA from in vivo and in vitro material. Of these, 5 distinguished a total of 13 polymorphisms common across 6 micropropagated plants. Apart from these 6 plants, the amplification products were monomorphic across all the micropropagated plants, the mother plant and 4 additional field-grown control plants. Our results show that RAPD markers can be used to gain rapid and precise information about genetic similarities or dissimilarities in micropropagation systems that might not be so easily evident from other commonly used techniques.     

Assessment of salt tolerance in Populus alba clones using chlorophyll fluorescence

Cuttings of five Populus alba clones (S18 F1-26, Al29 F8-35, J3 F1-4, GU1 F16-36, PO9 F21-88), Populus euphratica, and Populus×euramericana (I-214) were submitted during 45 d to regular watering with NaCl solutions of electrical conductivity of 7 and 14 dS m⁻¹. Chlorophyll a fluorescence in response to the salinity stress was assessed, using F₀ and F_v/F_m. Differences in reaction to the salt were found in P. alba clones, F₀ and F_v/F_m being the fluorescence parameters used to check out this stress. Minimal constant fluorescence of dark-adapted plants (F₀) showed a better correlation with the disease index exhibited by plants and also with salinity dose than the parameter F_v/F_m. Some of the P. alba clones showed the same behaviour, assessed through fluorescence parameters, as P. euphratica, which was previously defined as salt tolerant, while the rest exhibited the same characteristics as I-214, which was very sensitive.     

Reevaluation of the Life Cycle of Tuber magnatum

Tuber spp. are ectomycorrhizal ascomycetes that produce ascocarps known as truffles. Basic aspects of Tuber biology have yet to be fully elucidated. In particular, there are conflicting hypotheses concerning the mating system and the ploidy level of the mycorrhizal and truffle hyphae. We used polymorphic microsatellites to compare the allelic configurations of asci with those from the network of the surrounding hyphae in single Tuber magnatum truffles. We then used these truffles to inoculate host plants and evaluated the microsatellite configurations of the resulting mycorrhizal root tips. These analyses provide direct evidence that T. magnatum outcrosses and that its life cycle is predominantly haploid. In addition to its scientific significance, this basic understanding of the T. magnatum life cycle may have practical importance in developing strategies to obtain and select nursery-produced mycorrhizal plants as well as in the management of artificial plantations of this and other Tuber spp.     

Microbial and pigment profile of the reddish patch occurring within Tuber magnatum ascomata

Tuber magnatum Pico, the delectable white truffle, is the most prized truffle species. In this study, we examined the reddish pigmentation that frequently occurs in T. magnatum ascomata for the presence of pigment-producing bacteria.The inner part of the reddish-pigmented region of three T. magnatum ascomata collected in North–Central Italy was analysed. This reddish part was used to establish a bacterial culture collection and to extract the total genomic DNA in order to obtain a library of 16S rRNA genes representative of the bacterial community. The molecular approach revealed limited microbial diversity within the reddish-pigmented regions compared to the wider range of bacterial species commonly found at the same maturation stage and season in T. magnatum ascomata. The pigmented regions showed a prevalence of specific bacterial species belonging to α-, β- and γ- Proteobacteria, Actinobacteria and Firmicutes. From the tandem mass spectrometry analysis of the extracted pigment, four compounds were identified: i) bixin, ii) β-carotene, iii) cis-1-glycosyl-apo-8′- lycopene and iv) the fucoxanthin. Carotenoid producing species such as Microbacterium and Chryseobacterium emerged as the most likely cause of the peculiar reddish pigment production. Indeed, our findings suggest that the peculiar reddish pigment might be produced by these bacterial species.     

Production, turnover and mycorrhizal colonization of root systems of three Populus species grown under elevated CO2 (POPFACE)

A fast growing high density Populus plantation located in central Italy was exposed to elevated carbon dioxide for a period of three years. An elevated CO₂ treatment (550 ppm), of 200 ppm over ambient (350 ppm) was provided using a FACE technique. Standing root biomass, fine root turnover and mycorrhizal colonization of the following Populus species was examined: Populus alba L., Populus nigra L., Populus x euramericana Dode (Guinier). Elevated CO₂ increased belowground allocation of biomass in all three species examined, standing root biomass increased by 47–76% as a result of FACE treatment. Similarly, fine root biomass present in the soil increased by 35–84%. The FACE treatment resulted in 55% faster fine root turnover in P. alba and a 27% increase in turnover of roots of P. nigra and P. x euramericana. P. alba and P. nigra invested more root biomass into deeper soil horizon under elevated CO₂. Response of the mycorrhizal community to elevated CO₂ was more varied, the rate of infection increased only in P. alba for both ectomycorrhizal (EM) and arbuscular mycorrhizas (AM). The roots of P. nigra showed greater infection only by AM and the colonization of the root system of P. x euramericana was not affected by FACE treatment. The results suggest that elevated atmospheric CO₂ conditions induce greater belowground biomass investment, which could lead to accumulation of assimilated C in the soil profile. This may have implications for C sequestration and must be taken into account when considering long‐term C storage in the soil.     

Essential Elements as a Distinguishing Factor between Mycorrhizal Potentials of Two Cohabiting Truffle Species in Riparian Forest Habitat in Serbia

True truffles (Tuber sp.) that establish ectomycorrhizal symbiosis (ECM) with trees in the Mediterranean and temporal regions have species specific abilities to assimilate soil born elements. Suitable habitats are usually inhabited by few truffle species, while distinguishing their symbiotic potentials appeared very difficult. Two species that commonly inhabit riparian forests in Serbia are the most prized one, Tuber magnatum Pico (Piedmont white truffle) and not so highly valued Tuber brumale Vitt . In order to assess potential differences between their assimilation and accumulation abilities, the differences between contents of elements that may be the subjects of the symbiotic trade between the host plant and fungi were evaluated in accumulation target (ascocarps) and their source (the soil). Essential (K, Na, Ca, Mg, Fe, P, S, and Zn) and essential trace elements (Co, Cr, Cu, Mn, and Se) in truffles and soil samples were determined by means of inductively coupled plasma with optical emission spectrometry (ICP‐OES). Their concentrations (mg/kg) in ascocarps were in the range from 1.364±0.591 (Cr) to 10760.862±16.058 (K), while in soil ranged from 23.035±0.010 (Cr) to 20809.300±122.934 (Fe). Element accumulation potential (bioaccumulation factor) was calculated in the system truffle/soil. The statistical approaches were used for establishing the differences, while the possible differentiation between symbiotic potentials of two mycelia in the defined soil conditions was discussed.     

Transformation of elite white poplar (Populus alba L.) cv. ' Villafranca' and evaluation of herbicide resistance

 Transgenic white poplar plants (Populus alba L.) expressing the nptII gene and the bar gene from Streptomyces hygroscopicus have been produced using Agrobacterium tumefaciens-mediated gene transfer. Eleven kanamycin-resistant plant lines were obtained with a transformation frequency of 7%. Successful genetic transformation was confirmed by Southern and northern analyses. The level of resistance to the commercial preparation of phosphinothricin (Basta; Roussel-Hoechst Agrovet) was evaluated by in vitro and in vivo assays. Using in vitro selective conditions for phosphinothricin, only plantlets from four kanamycin-resistant independent lines remained green and continued to grow and root. After transfer to the growth chamber, all selected transgenic lines were shown to be completely resistant to the herbicide Basta with doses equivalent to 6 l ha^–1 (normal field dosage) and were tolerant at concentration of 12 l ha^–1. This is the first report describing the genetic transformation of a P. alba clonal cultivar of commercial interest with a gene of agronomic value. Received: 12 June 1999 / Revision received: 6 March 2000 / Accepted: 7 March 2000     

Effects of a mixed-isolate mycorrhizal inoculum on the potato – potato cyst nematode interaction

Inoculation of microplants of potato cv. Golden Wonder with Vaminoc, a mycorrhizal inoculum of three arbuscular mycorrhizal fungi (Glomus spp.), resulted in an increase in in‐sand hatch of Globodera pallida, but not G. rostochiensis, within 2 weeks. By this time, mycorrhized plants also supported a larger number of feeding nematodes of both PCN species (50% higher for G. rostochiensis) than did non‐mycorrhized plants, with a higher proportion of the G. pallida population being fertilised females than for G. rostochiensis. After 12 weeks, the multiplication rate of G. rostochiensis on mycorrhized plants was significantly greater than on non‐mycorrhized plants, whereas no such difference was observed for G. pallida. The principal component of PCN multiplication affected by mycorrhization was increased cyst number per plant from 6 to 12 weeks. Over this period, there was no increase in cyst number per plant for either PCN species on non‐mycorrhized plants, whereas the value increased on mycorrhized plants for both G. rostochiensis (by almost 200%) and G. pallida (57%). Mycorrhization resulted in significant increases in the root and shoot dry weights of plants grown in the absence of PCN. Although mycorrhized plants carried a larger PCN burden than non‐mycorrhized plants when grown on PCN‐infested medium, as a result of the increased PCN multiplication rate, they produced larger root systems than did nonmycorrhized plants, suggesting increased tolerance to PCN of the mycorrhized plants, particularly to G. rostochiensis. Of morphological characters investigated in the absence of PCN, only stem height (increased) was significantly affected by mycorrhization. Colonisation by mycorrhizal fungi resulted in increased tuber yield both in the absence (significant increase) and presence (non significant) of PCN, as a result of increased tuber number per plant. These results are discussed in the light of the possible use of AMF as part of an integrated PCN management plan.     

Infectivity and effectiveness of Glomus intraradices on micropropagated plants

Colonization by Glomus intraradices takes place very early within the root system of micropropagated plantlets of strawberry (var. avanta, elsanta), raspberry (var. himboqueen, Zeva I), and hortensia (var. leuchtfeuer). The arbuscular mycorrhizal fungus (AMF) did not colonize roots of the different hosts to the same extent, and considerable differences were observed between the varieties. The results reported here confirm that endomycorrhizal root colonization is affected by the host-fungus combination. The effects ranged from mutualistic (hortensia), through neutral (strawberry var. avanta, raspberry var. Zeva I) to negative (raspberry var. himboqueen and strawberry var. elsanta). Non-mycorrhized (control) plants of strawberry produced more runners than mycorrhized plants under controlled growth conditions (phytotron). Transfer of the potted plants to the field resulted in drastic alterations in overall growth and development within 4 weeks. Mycorrhized plants became healthy, and mycorrhized strawberry plants produced many stout runners. The number of the runners and their biomass were almost the same (var. avanta) and treated plants produced even more runners than the controls (var. elsanta). The authors have demonstrated the need to determine the specific effects of each species of AMF on individual prospective host plants prior to their utilization in the micropagation of plantlets.     

Effects of 3,4-dihydroxybenzoic acid on tobacco (Nicotiana tabacum L.) cultured in vitro. Growth regulation in callus and organ cultures

ABSTRACT

The influence of 3,4-dihydroxybenzoic acid (protocatechuic acid), a naturally occurring benzoic acid derivative, on tobacco (Nicotiana tabacum L.) cell and tissue cultures was examined. The response to 0.1, 10 and 1000 µM 3,4-dihydroxybenzoic acid was tested with regards to cell proliferation in leaf explants, callus growth and shoot formation. Effects on shoot and root growth in micropropagated plants were also analysed. The highest concentration of 3,4-dihydroxybenzoic acid strongly inhibited the proliferation of leaf tissues, callus growth, shoot regeneration and root growth in micropropagated plants. On the contrary, the lowest concentration (0.1 µM) showed auxin-like activity by stimulating cell dedifferentiation, callus induction and rooting of leaf tissues. The presence of auxins and cytokinins in the media contrasted 3,4-dihydroxybenzoic acid inhibition of callus growth at all tested concentrations.     

Osservazioni sulla microflora fungina di ceppaie di abete bianco

Abstract

Mycoflora of Silver fir (Abies alba Mill.) stumps. - This work is a first contribution to the knowledge of the mycoflora colonizing Silver fir (Abies alba Mill.) stumps in Italy.

Autochthonous fungal species were isolated from the wood of A. alba stumps from three different sites in the Tuscan Apennines.

Frequency values for the isolated species and taxonomic groups were calculated.

Deuteromycetes (57–82.5%) occurred more frequently than basidiomycetes (5–36%), phycomycetes (0.7–11.6%) and ascomycetes (0–1%). The most common genera were found to be: Aleurodiscus, Penicillium, Phellinus, Phialophora, Scytalidium, Sistotrema and Trichoderma.

An effective method of isolation from wood is also described.     

Micropropagation of Tectona grandis: Assessment of Genetic Fidelity

Random amplified polymorphic DNA (RAPD) markers were used to analyze genetic fidelity of micropropagated teak (Tectona grandis L.) clones with respect to subcultural passage. Of the twenty primers screened, no variation in RAPD profiles was noticed in the in vitro clones of fifth, tenth, fifteenth and twentieth passage in comparison to the in vivo mother plants. Only one micropropagated plant of twenty-fifth subcultural passage, however, differed from the in vivo ones. It revealed the appearance of a new polymorphic DNA fragment (molecular mass 379 kb) in case of primer OPB-08. This primer, manifesting detectable variation, may be utilized as a diagnostic marker for assessing genetic fidelity of micropropagted teak plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic characterization and relationships of Populus alba,P. tremula,and P. x canescens,and their clones

Summary Isozyme analysis was conducted on individuals of Populus alba L., P. tremula L., and P. × canescens Smith to genetically characterize and differentiate species, hybrids, and individuals, and to determine genetic relationships among them. Thirty gene loci, with 71 alleles, coding for 15 enzymes were observed. Individuals could be identified on the basis of their multilocus genotypes. There were 21 unique multilocus genotypes among 23 P. alba clones. Five P. alba clones from Canada were genetically distinct from each other. Each of the 18 P. tremula and 15 P. × canescens clones had unique multilocus genotypes. Thirteen clones had a unique genotype at a single locus. Percentage of polymorphic loci, average number of alleles per locus, and mean observed heterozygosity were, respectively, 50.0, 1.86, and 0.085 in P. alba, 51.7, 1.66, and 0.096 in P. tremula, and 51.7, 1.86, and 0.157 in P. × canescens. Populus alba and P. tremula were genetically distinct from each other and could be distinguished by mutually exclusive alleles at Aco-3, P. tremula-specific gene Mdh-3, and allele frequency differences at 6 loci. Populus × canescens had allele contributions of P. alba and P. tremula. However, their allele frequencies were closer to those of P. alba than being truly intermediate. The mean genetic identity was 0.749 between P. alba and P. tremula, 0.987 between P. alba and P. × canescens, and 0.817 between P. tremula and P. × canescens. Canonical discriminant analysis of multilocus genotypes separated P. alba, P. tremula, and P. × canescens into three distinct groups and portrayed similar interspecific relationship as above. Our results suggested that the putative P. × canescens individuals consisted of a mixture of F₁ hybrids of P. alba and P. tremula and their backcrosses to P. alba.Presently with the University of Alberta, and BioGenetica Inc., P.O. Box 8261, Edmonton, Alberta, Canada T6H 4P1     

Genetic and Phylogeographic Structures of the Symbiotic Fungus Tuber magnatum

The quality and market price of truffles vary with the species and, traditionally, the place of origin. The premium species Tuber magnatum produces white truffles and has a patchy distribution restricted to Italy and some Balkan areas. We used polymorphic microsatellites to evaluate 316 specimens grouped into 26 populations sampled across the species' geographic range to determine if natural populations of T. magnatum are genetically differentiated. We found that the southernmost and the northwesternmost populations were significantly differentiated from the rest of the populations. The simple sequence repeat data also could be used to make inferences about the postglacial T. magnatum expansion pattern. This study is the first to identify a genetic and phylogeographic structure in T. magnatum. The presence of a genetic structure can be of practical interest in tracing truffle populations according to their geographic origin for marketing strategies. Evidence for extensive outcrossing in field populations of T. magnatum also is provided for the first time.     

Assessment of ectomycorrhizal fungal communities in the natural habitats of Tuber magnatum (Ascomycota, Pezizales)

The ectomycorrhizal (ECM) fungal communities of four natural Tuber magnatum truffle grounds, located in different Italian regions (Abruzzo, Emilia-Romagna, Molise, and Tuscany), were studied. The main objective of this study was to characterize and compare the ECM fungal communities in the different regions and in productive (where T. magnatum ascomata were found) and nonproductive points. More than 8,000 (8,100) colonized root tips were counted in 73 soil cores, and 129 operational taxonomic units were identified using morphological and molecular methods. Although the composition of the ECM fungal communities studied varied, we were able to highlight some common characteristics. The most plentiful ECM fungal taxa belong to the Thelephoraceae and Sebacinaceae families followed by Inocybaceae and Russulaceae. Although several ectomycorrhizas belonging to Tuber genus were identified, no T. magnatum ectomycorrhizas were found. The putative ecological significance of some species is discussed.     

An improved micropropagation of <Emphasis Type="Italic">Eclipta alba</Emphasis> by in vitro priming with chlorocholine chloride

An efficient method of micropropagation for Eclipta alba from young nodal axils of shoot tip explants has been developed by giving special attention to ‘priming’ in vitro plantlets in view of increasing their hardening ability after transplantation ex vitro. Among 3 cytokinins—BAP, kinetin and TDZ, BAP was found most effective in inducing and proliferating adventitious shoots. The highest frequency of responding explants (100%) and maximum number of shoots (23.0) per explant were obtained after 60 days culture on MS medium containing 8.8 μM BAP. Cent percent shoots developed roots directly from shoot base when transferred to growth regulator-free MS medium. For priming E. alba microshoots, 6.3 μM of chlorocholine chloride (CCC) was found most effective. The major changes observed in 30 days old treated shoots were, production of increased number of root, elevation of chlorophyll level in leaves and increase in plant biomass. Furthermore, arrested undesirable shoot elongation made the plants sturdier and more suitable for acclimatization. The primed micropropagated E. alba plants were healthy and survived by higher frequency (100%) in soil in comparison to the non-treated plants (84% survival).     

Transformation of white poplar (Populus alba L.) with a novel Arabidopsis thaliana cysteine proteinase inhibitor and analysis of insect pest resistance

Transgenic white poplar (Populus alba L.) plants expressing a novel Arabidopsis thaliana cysteine proteinase inhibitor (Atcys) gene have been produced using Agrobacterium tumefaciens-mediated gene transfer. Internodal stem segments of cv. Villafranca were co-cultivated with the EHA105 pBI-Atcys A. tumefaciens strain. Sixteen putative transgenic plant lines were regenerated from different calli with a transformation efficiency of 11%. The integration and expression of the cysteine proteinase inhibitor (Atcys) gene into the plant genome was confirmed by Southern and northern blot analyses. Papain inhibitory activity was detected in poplar transgenic tissues by means of a specific in vitro assay. Such activity was sufficient to inhibit most of the digestive proteinase activity of chrysomelid beetle (Chrysomela populi L.) and confer resistance to C. populi larvae on selected transgenic plants. A close correspondence between the inhibition of papain and resistance to poplar leaf beetle was observed in all tested transgenic lines. Our results indicate that Atcys could be succesfully employed in breeding programmes aimed at the selection of new poplar genotypes resistant to major insect pests.     

Rapid clonal propagation of Saccharum officinarum L. Vars. CO-6907 and CO-86249 and to assess the genetic uniformity through molecular markers

Abstract

An efficient protocol was developed for in vitro clonal propagation of Saccharum officinarum Vars. CO-6907 and CO-86249 through axillary meristem culture. Maximum meristem elongation was achieved on Murashige and Skoog's (MS) medium supplemented with 0.5 mg/L 6-benzyladenine (BA) and 0.5 mg/L kinetin (Kn) within 15 days of culture. Multiple shoots were induced from meristems on MS basal medium supplemented with 1.0 mg/L BA, 0.5 mg/L Kn, 0.25 mg/L 1-napthaleneacetic acid (NAA) and 3% (w/v) sucrose. Addition of 0.1–0.25 mg/L gibberellic acid into the multiplication medium found the better shoot elongation. Repeated subculture on multiplication medium induces higher rate of shoot multiplication. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 1.0–2.0 mg/L NAA or indole-3-butyric acid and 6% (w/v) sucrose. While either decreasing or increasing of sucrose concentration in the rooting medium, the percentage of rooting was reduced. Maximum percentage of rooting was achieved on medium having 2.0 mg/L NAA with 6% (w/v) sucrose. About 80% of micropropagated plantlets were hardened in the greenhouse and successfully established in the soil. Random Amplified Polymorphic DNA marker was used to detect the variability among the micropropagated plants developed through in vitro. The results showed that there was no polymorphism among the micropropagated plants. This study will help for propagation of quality planting material of high-yielding variety of sugarcane for commercialization.