Two new species of spotted Hypancistrus from the Rio Negro drainage (Loricariidae,Hypostominae)

Two new species, Hypancistrus phantasma and Hypancistrus margaritatus, are described based on material from the Rio Negro drainage. Both species are distinguished from congeners by unique color patterns. Hypancistrus phantasma is described from the Rio Uaupes and differs from congeners by having a tan body with small dark spots (vs. dark with light spots or with saddles or stripes). Hypancistrus margaritatus is described from the Takutu River and differs from congeners by having densely-packed light spots on a dark brown background, with spots about the size of the nasal aperture (vs. sparse light spots either smaller or larger than the nasal aperture, or brown to black spots, saddles, or stripes).     

Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) eggs as alternative food for rearing of lady beetles Eriopis connexa (Germar) (Coleoptera: Coccinellidae)

Eriopis connexa (Germar) (Coleoptera: Coccinellidae) is an important predator with potential for biological control of insect pests. This research evaluated the development of E. connexa larvae fed on fresh eggs of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) without (T1) or with (T2) scales or one-day (T3) or six-month (T4) frozen, or newly-hatched larvae of S. frugiperda (T5). The percentage of E. connexa adults was higher when larvae feeding on fresh S. frugiperda eggs with or without scales, or one-day frozen eggs of this prey and lower with eggs of this Lepidoptera after frozen for six months or with newly-hatched larvae of S. frugiperda. Duration of the larval period of E. connexa was 15.7, 15.8, 16.0, 17.6, and 17.3 days, respectively, with these diets. The high survival of E. connexa fed with eggs of S. frugiperda shows the potential use of this prey in the laboratory to maintain this natural enemy.     

Interaction between entomopathogenic nematodes and entomopathogenic fungi applied to third instar southern masked chafer white grubs,Cyclocephala lurida (Coleoptera: Scarabaeidae), under laboratory and greenhouse conditions

Four entomopathogenic nematode (EPN) species (Heterorhabditis bacteriophora Poinar, Heterorhabditis megidis Poinar, Jackson & Klein, Steinernema feltiae Filipjev and Steinernema riobrave Cabanillas, Poinar & Raulston) were tested for virulence against 3^rd instar southern masked chafer white grubs, Cyclocephala lurida Bland. H. bacteriophora and H. megidis, being the most virulent, were selected to evaluate the interaction with an entomopathogenic fungus (EPF), Beauveria bassiana (Balsamo) Vuillemin strain GHA or Metarhizium anisopliae (Metsch.) Sorokin strain F-52, under laboratory and greenhouse conditions. Nematodes and fungi were either applied alone or in combination, with nematodes added to fungi at different times. When applied alone, B. bassiana and M. anisopliae did not reduce grub numbers. Under laboratory conditions, additive interactions were found between H. megidis and B. bassiana, and between H. bacteriophora and B. bassiana or M. anisopliae in most combinations against chafer grubs; a few treatments showed synergism or antagonism. The combined effect did not differ significantly for nematode and fungal applications made simultaneously or at different times. Nematode infection and infective juveniles (IJs) production in grub carcasses were not significantly affected by the presence of a fungus. Efficacies of H. bacteriophora and M. anisopliae were affected by temperature, with grub mortality increasing at higher temperatures. Under greenhouse conditions, additive or synergistic interaction was found between H. bacteriophora and B. bassiana or M. anisopliae in different formulations in simultaneous applications or when the nematode was applied 4 weeks after the fungi, except between B. bassiana ES and H. bacteriophora. The impact of H. bacteriophora alone or in combination with M. anisopliae or B. bassiana on 3^rd instar C. lurida was comparable to that of an imidacloprid insecticide used as curative applications. More virulent fungal strains or species may be required to achieve a stronger interaction with nematodes in the management of C. lurida.     

Studies on the specificity of killing of intracellular pathogens by macrophages.

To determine whether macrophages can discriminate in an immunologically specific manner between the intracellular pathogens which they inhibit or kill, unelicited peritoneal macrophages from mice infected with either of two related but antigenically dissimilar protozoa were challenged with these protozoa in vitro. Experimental conditions were varied in an attempt to establish a state in vivo in which macrophage specificity might be demonstrated. No differences could be discerned between the ability of macrophages from three different strains of mice infected with the protozoa to kill Besnoitia and Toxoplasma. The effect of macrophages on Toxoplasma as compared with Besnoitia did not evolve or vary during development, expression, or decline of an immune response, i.e., with varying times after infection of mice as well as with varying times after treatment of mice with irradiated Toxoplasma. The route of infection could not be shown to confer specificity on macrophages, as subcutaneous and intraperitoneal inoculation of Toxoplasma did not lead to differential ability of macrophages to inhibit or kill the protozoa. The different strains of protozoa used for infection of mice did not affect the ability of peritoneal macrophages from Besnoitia- and Toxoplasma-infected mice to inhibit multiplication of or kill Besnoitia and Toxoplasma comparably in vitro. Peritoneal macrophages of mice treated with Corynebacterium parvum kill both organisms efficiently. These macrophages were employed to determine whether stimulation of macrophages by treatment of mice with a substance unrelated to the protozoa would produce activated macrophages. Uninfected mice and mice infected with either Besnoitia or Toxoplasma were challenged with varying doses of the protozoa in parallel with examination of macrophages from the same groups of mice in vitro to determine whether the presence of stimulated macrophages in the peritoneal cavity was necessary for protection against Toxoplasma and Besnoitia, and if so if their presence was sufficient for protection. Only mice with activated peritoneal macrophages were protected. However, protection was greater when the primary infection was with the same organisms used for challenge at a time when macrophages inhibited or killed both protozoa efficiently in vitro. The possible role of other effector cells, subpopulations of macrophages of different functional abilities in various sites, and antibody or other lymphocyte products acting in concert with macrophages as factors which may explain the differences observed between in vivo protection and in vitro capacity to inhibit or kill the protozoa are discussed.     

Use of Entomopathogenic Nematodes to Suppress Meloidogyne incognita on Greenhouse Tomatoes

Tomato seedlings in a growth chamber were inoculated with 150 Meloidogyne incognita eggs and 25 infective juveniles (IJ)/cm² of Steinernema feltiae, S. riobrave, or Heterorhabditis bacteriophora. With the exception of seedling roots treated with H. bacteriophora, all seedlings treated with entomopathogenic nematodes had fewer M. incognita juveniles inside roots and produced fewer eggs than the control seedlings. Tomato plants in the greenhouse were infested with 4,000 M. incognita eggs and treated 2 weeks before, 1 week before, at the same time, 1 week after, or 2 weeks after with 25 or 125 IJ/cm² of S. feltiae, S. riobrave, or H. bacteriophora. Plants with pre- and post-infestation applications of S. feltiae or S. riobrave suppressed M. incognita. Plants treated with H. bacteriophora 1 week before and at the time of infestation suppressed M. incognita. Increasing the rate of H. bacteriophora and S. feltiae from 25 to 125 IJ/cm² improved M. incognita suppression.     

Influence of Concomitant Pratylenchus brachyurus and Meloidogyne spp. on Root Penetration and Population Dynamics

Populations of Pratylenchus brachyurus on cotton were increased significantly in the presence of either Meloidogyne incognita or M. arenaria.This occurred with either simultaneous inoculation or prior invasion by M. incognita. P. brachyurus penetrated cotton roots previously invaded by, or simultaneously inoculated with, M. incognita, as well as, or better than, in the absence of M. incognita. Prior invasion by M. incognita, however, suppressed P. brachyurus populations on tomato, while it had no effect on alfalfa and tobacco. Populations of M. incognita on cotton were generally inhibited by the presence of P. brachyurus. Simultaneous inoculation with, or previous invasion by, P. brachyurus also inhibited root penetration by M. incognita. These findings emphasize the importance of host susceptibility in the study of concomitant nematode populations.     

Polymorphisms in Fibronectin Binding Proteins A and B among Staphylococcus aureus Bloodstream Isolates Are Not Associated with Arthroplasty Infection

Background

Nonsynonymous single nucleotide polymorphisms (SNPs) in fibronectin binding protein A (fnbA) of Staphylococcus aureus are associated with cardiac device infections. However, the role of fnbA SNPs in S. aureus arthroplasty infection is unknown.

Methods

Bloodstream S. aureus isolates from a derivation cohort of patients at a single U.S. medical center with S. aureus bacteremia (SAB) and prosthetic hip or knee arthroplasties that were infected (PJI, n = 27) or uninfected (PJU, n = 43) underwent sequencing of fnbA and fnbB. A validation cohort of S. aureus bloodstream PJI (n = 12) and PJU (n = 58) isolates from Germany also underwent fnbA and fnbB sequencing.

Results

Overall, none of the individual fnbA or fnbB SNPs were significantly associated with the PJI or PJU clinical groups within the derivation cohort. Similarly, none of the individual fnbA or fnbB SNPs were associated with PJI or PJU when the analysis was restricted to patients with either early SAB (i.e., bacteremia occurring <1 year after placement or manipulation of prostheses) or late SAB (i.e., bacteremia >1 year after placement or manipulation of prostheses).

Conclusions

In contrast to cardiac device infections, there is no association between nonsynonymous SNPs in fnbA or fnbB of bloodstream S. aureus isolates and arthroplasty infection. These results suggest that initial steps leading to S. aureus infection of cardiovascular and orthopedic prostheses may arise by distinct processes.     

Plasmid-Encoded Pgp5 Is a Significant Contributor to Chlamydia muridarum Induction of Hydrosalpinx

We have previously shown that the plasmid-encoded Pgp3 is a major virulence factor for C. muridarum induction of hydrosalpinx. We now report that Pgp5 also plays a significant role in the development of hydrosalpinx following C. muridarum induction. Pgp5 deficiency was introduced via either in-frame deletion (CM-Δpgp5) or premature stop codon installation (CM-pgp5S). Mice infected with either CM-Δpgp5 or CM-pgp5S developed hydrosalpinges at significantly reduced levels with an incidence rate of <40% and a mean severity score of 2 or less. In contrast, 80% or more mice developed hydrosalpinx with a severity score of >3 when mice were infected with Pgp5-sufficient C. muridarum (plasmid-competent wild type or plasmid-free C. muridarum transformed with a full plasmid or depleted of pgp7 gene). The attenuated pathogenicity of the Pgp5-deficient C. muridarum correlated with a significantly reduced level of ascending infection in the oviduct tissue despite the similar overall shedding courses between mice infected with Pgp5-deficeint versus sufficient C. muridarum. Furthermore, in the oviducts of mice infected with Pgp5-deficient C. muridarum, significantly lower levels of inflammatory cell infiltration and cytokine production were detected. Thus, Pgp5 is a significant plasmid-encoded virulence factor for C. muridarum pathogenicity in the upper genital tract.     

Major trends of evolution in palms

From the diversity found among palms the following evolutionary trends are suggested:habit: from sympodial to monopodial;size: from moderate toward large and also toward small;stem: from unbranched to dichotomously branched, from little to much sclerenchyma, from short to elongate internodes;leaf: from an undivided eophyll to a palmate, costapalmate, pinnately ribbed or pinnate blade; from undivided and plicate to divided along the adaxial rib (“induplicate”) or along the abaxial rib (“reduplicate”); from pinnate to bipinnate or to pinnae onceor twicedivided longitudinally; from sheath split opposite the petiole to sheath tubular; from marcescent to deciduous; from central vascular bundles of the petiole with a single phloem strand to two phloem strands;inflorescence units: from moderately branched to spicate or less frequently to more diffusely branched, from one unit per leaf axil to more than one per axil, from among the leaves to below them or to above them in a compound terminal inflorescence, from pleonanthic to hapaxanthic;prophyll: from completely to incompletely encircling the peduncle, from incompletely to completely sheathing in bud;bracts: from conspicuous to small or absent at maturity, first peduncular bract from tubular and open at the apex to completely enclosing the inflorescence in bud, and then from ungrooved to deeply plicate;flower arrangement: from solitary, pedicellate, bracteolate flowers to a sympodial cincinnus of 2 or 3 or more, or to a short monopodial axis of 2–4 or more;bracteoles: from sheathing and prophyllate to completely closed or to incompletely developed or absent;flowers: from bisexual to unisexual, then associated with polygamy or monoecism to dioecism;perianth: from trimery to dimery or tetramery to decamery or to reduced and monochlamydeous;sepals: from distinct and imbricate to connate or separated;petals: from distinct and imbricate to valvate, or strongly imbricate, or connate; from small and ovate to large and variously shaped, or to small;stamens: from 6 to 3 or to more than 6 (to 950+);filaments: from relatively slender and distinct to broad and thick, and often connate or adnate to the perianth or both;staminodes: from stamenlike with abortive anthers only, to short teeth, or to a cupule at the base of the ovary, or to absent;pollen: from monosulcate to trichotomosulcate to dicolpate to monocolpate, diporate, or triporate;gynoecium: from apocarpous to syncarpous, from thin walls to thick, variously specialized walls;carpels or locules: from 3 to 2-1 or to 4–10;ovules: from moderate to small or to large, from anatropous to hemianatropous to campylotropous to orthotropous;pistillode: from only slightly modified from the gynoecium to vestigial or lacking or rarely to prominent;fruit: from fleshy to dry and fibrous;endocarp: from little differentiated or thin, to thick and hard, and sometimes with a pore or operculum over the embryo;seed: from moderate to small or to very large, from entire to dissected, bilobed, or perforate;endosperm: from homogeneous to invaginated or ruminate;germination: from remotetubular or -ligular to adjacent-ligular;chromosome complement: fromn = 18 ton = 17, 16, 15, 14, 13.     

MicroRNA-196a and -196b as Potential Biomarkers for the Early Detection of Familial Pancreatic Cancer

Screening programs are recommended for individuals at risk (IAR) from families with familial pancreatic cancer (FPC). However, reliable imaging methods or biomarkers for early diagnosis of pancreatic ductal adenocarcinoma (PC) or its precursor lesions are still lacking. The ability of circulating microRNAs (miRNAs) to discriminate multifocal high-grade precursor lesions or PC from normal was examined. The presence of miRNA-21, -155, -196a, -196b and -210 was analyzed in the serum of transgenic KPC mice to test their ability to distinguish mice with different grades of pancreatic intraepithelial neoplasia (mPanIN1–3) or PC from control mice. Serum levels of miR-196a and -196b were significantly higher in mice with PanIN2/3 lesions (n = 10) or PC (n = 8) as compared to control mice (n = 10) or mice with PanIN1 lesions (n = 10; P = .01). In humans, miR-196a and -196b were also diagnostic. Patients with PC, sporadic (n = 9) or hereditary (n = 10), and IAR with multifocal PanIN2/3 lesions (n = 5) had significantly higher serum levels than patients with neuroendocrine pancreatic tumors (n = 10) or chronic pancreatitis (n = 10), IAR with PanIN1 or no PanIN lesions (n = 5), and healthy controls (n = 10). The combination of both miR-196a and -196b reached a sensitivity of 1 and specificity of 0.9 (area under the curve = 0.99) to diagnose PC or high-grade PanIN lesions. In addition, preoperative elevated serum levels of miR-196a and -196b in patients with PC or multifocal PanIN2/3 lesions dropped to normal after potential curative resection. The combination of miR-196a and -196b may be a promising biomarker test for the screening of IAR for FPC.     

Molecular and phenotypic characterization of seedling and adult plant leaf rust resistance in a world wheat collection

Genetic resistance is the most effective approach to managing wheat leaf rust. The aim of this study was to characterize seedling and adult plant leaf rust resistance of a world wheat collection. Using controlled inoculation with ten races of Puccinia triticina, 14 seedling resistance genes were determined or postulated to be present in the collection. Lr1, Lr3, Lr10 and Lr20 were the most prevalent genes around the world while Lr9, Lr14b, Lr3ka and/or Lr30 and Lr26 were rare. To confirm some gene postulations, the collection was screened with gene-specific molecular markers for Lr1, Lr10, Lr21 and Lr34. Although possessing the Lr1 and/or Lr10 gene-specific marker, 51 accessions showed unexpected high infection types to P. triticina race BBBD. The collection was tested in the field, where rust resistance ranged from nearly immune or highly resistant with severity of 1 % and resistant host response to highly susceptible with severity of 84 % and susceptible host response. The majority of the accessions possessing the adult plant resistance (APR) gene Lr34 had a maximum rust severity of 0–35 %, similar to or better than accession RL6058, a Thatcher-Lr34 near-isogenic line. Many accessions displayed an immune response or a high level of resistance under field conditions, likely as a result of synergy between APR genes or between APR and seedling resistance genes. However, accessions with three or more seedling resistance genes had an overall lower field severity than those with two or fewer. Immune or highly resistant accessions are potential sources for improvement of leaf rust resistance. In addition, some lines were postulated to have known but unidentified genes/alleles or novel genes, also constituting potentially important sources of novel resistance.     

Rapid and accurate species and genomic species identification and exhaustive population diversity assessment of Agrobacterium spp. using recA-based PCR

Agrobacteria are common soil bacteria that interact with plants as commensals, plant growth promoting rhizobacteria or alternatively as pathogens. Indigenous agrobacterial populations are composites, generally with several species and/or genomic species and several strains per species. We thus developed a recA-based PCR approach to accurately identify and specifically detect agrobacteria at various taxonomic levels. Specific primers were designed for all species and/or genomic species of Agrobacterium presently known, including 11 genomic species of the Agrobacterium tumefaciens complex (G1-G9, G13 and G14, among which only G2, G4, G8 and G14 still received a Latin epithet: pusense, radiobacter, fabrum and nepotum, respectively), A. larrymoorei, A. rubi, R. skierniewicense, A. sp. 1650, and A. vitis, and for the close relative Allorhizobium undicola. Specific primers were also designed for superior taxa, Agrobacterium spp. and Rhizobiaceace. Primer specificities were assessed with target and non-target pure culture DNAs as well as with DNAs extracted from composite agrobacterial communities. In addition, we showed that the amplicon cloning-sequencing approach used with Agrobacterium-specific or Rhizobiaceae-specific primers is a way to assess the agrobacterial diversity of an indigenous agrobacterial population. Hence, the agrobacterium-specific primers designed in the present study enabled the first accurate and rapid identification of all species and/or genomic species of Agrobacterium, as well as their direct detection in environmental samples.     

Maternal and neonate diatom diets impair development and sex differentiation in the copepod Temora stylifera

Development and sex differentiation in the copepod Temora stylifera was studied in the presence of maternal and larval diets of the diatoms Thalassiosira rotula and Skeletonema marinoi, either provided alone or supplemented with the control dinoflagellate Prorocentrum minimum. Both diatoms had deleterious effects on growth compared to the control when used as pure diets, inducing very low or even zero survival from hatching to adulthood. This effect was deleted when the diet was supplemented with a good food (P. minimum) only in the case of T. rotula. By contrast, with a maternal or larval diet of S. marinoi, nauplii did not pass metamorphosis even when this alga was mixed with P. minimum. Arrested development was not due to lack of feeding since early and late nauplii (NII and NV) ingested all three algae at similar rates when used as single diets, and did not show any preference when the algae were offered as mixtures. Since mortality rates with a mixed diet of S. marinoi + P. minimum were similar or even higher than those obtained with a single diet of S. marinoi, we suggest that this diatom is more toxic than nutritionally deficient for T. stlyfera development. No males were produced in cohorts reared on pure diatom diets or with a mixture of S. marinoi + P. minimum, and intermediate male:female sex ratios were obtained with the mixed T. rotula + P. minimum diet. Possibly some diatoms produce compounds such as oxylipins or new molecules that alter sex differentiation in T. stylifera.     

Reduction methods for logical control networks

In this paper some earlier stated theorems on logical control networks are proved. It is proved that a cascade of elements is equivalent to a single positive or negative element with N, number of negative elements, respectively even or odd. A loop of elements may show a cyclic behaviour with N odd or may show two possible stationary states with N even. A grafted cascade on a loop destroys the typical behaviour of the loop if the cascade is a negative chain grafted with an AND or a positive chain grafted with an OR connection; the system then admits only one stable state. Grafting a negative chain with an OR or a positive chain with an AND connection leaves the general behaviour of the loop unaffected. The detailed behaviour of two interconnected loops is extensively described. Finally it is indicated that although a complex network can be formalized as a reduced graph, its topological properties cannot always predict the final possible behaviour.     

Effects of Management Practices on Nematode and Fungi Populations and Okra Yield

Okra was grown in field plots of Tifton loamy sand naturally infested with the nematodes Meloidogyne incognita and Criconemoides ornalus and the pathogenic fungi Fusarium oxysporum, F. solani, F. roseum, and Pythium spp. Plots were treated with various soil pesticides and left exposed or covered with biodegradable paper film mulch under trickle irrigation. Soil was assayed for nematodes and fungi, and plant roots were examined for root-rot and insect damage. Fewer nematodes and fungi generally were recovered from soil treated with DD-MENCS (with and without film mulch) or methyl bromide-chloropicrin (2:1) (MBC) and film mulch than from nontreated soil. Funfigation with DD-MENCS or MBC suppressed populations of M. incognita, C. ornatus, F. oxysporum, F. solani, F. roseum, and Pythium spp. Ethoprop (alone or combined with other pesticides), sodium azide, and chloroneb were less effective than DD-MENCS and MBC. Plant growth anti yield were greatest when nematodes and pathogenic fungi were controlled. Yield was increased 3-fold by DD-MENCS + film mulch or MBC + film mulch in comparison with the average yield of okra produced in Georgia. The root-knot nematode-Fusarium wilt complex was most severe in nonfuntigated soil.     

Effects of Soil Temperatures and Inoculum Levels of Meloidogyne incognita and Rhizoctonia solani on Seedling Disease of Cotton

Soreshin of cotton was more severe from combined infections of Rhizoctonia solani and Meloidogyne incognita than from either organism alone, when both critical soil temperature and inoculum concentrations were present. Optimum soil temperatures for disease development from combined infections were 18 and 21 C. Either 2,500 or 5,000 M. incognita larvae per plant, combined with R. solani, increased seedling disease severity over that caused by R. solani alone. When 100 or 500 larvae per plant were added with R. solani, disease severity did not change. Disease severity increased with the highest level of R. solani inoculum either alone or combined with M. incognita.     

黄土高原樟子松和落叶松与其他树种枯落叶混合分解对土壤的影响

通过采集树木枯落叶与土壤进行室内混合分解培养试验,研究了黄土高原常见的樟子松和落叶松与其他树种枯落叶混合分解对土壤性质的影响及存在的相互作用,从而为不同树木种间关系的探索和该地区人工纯林的混交改造提供科学指导。结果表明:12种枯落叶单一分解均明显提高了土壤脲酶(54%—110%)、脱氢酶(85%—288%)和磷酸酶(81%—301%)活性以及有机质(29%—55%)和碱解N(12%—49%)含量,但对土壤速效P含量和CEC的影响存在较大差异。综合而言,樟子松分别与白桦、刺槐、白榆、柠条和落叶松枯落叶混合分解在对土壤性质的影响中存在相互促进作用,而分别与小叶杨、沙棘、紫穗槐、侧柏和辽东栎枯落叶混合分解在对土壤性质的影响中存在相互抑制作用;落叶松分别与刺槐、白桦、小叶杨和紫穗槐枯落叶混合分解在对土壤性质的影响中存在相互促进作用,而分别与柠条、侧柏、辽东栎、沙棘、油松和白榆枯落叶混合分解在对土壤性质的影响中存在相互抑制作用。     

Survival and Reproduction of Some Nematodes as Affected by Muck and Organic Acids

Fulvic, humic, acetic, N-bulyric, formic, lactic, and propionic acids were inhibitory to the survival or reproduction of Aphelenchus avenae, Aphelenchoides goodeyi, Helicotylenchus pseudorobustus, Meloidogyne hapla or Xiphinema americanum. Reproduction of H. pseudorobustus and M. hapla significantly increased with increasing amounts of muck added to sand, and with the initial amount of nematode inoculum. All acids except humic and fulvic were lethal, in vitro, to all nematode species tested. When A. goodeyi was treated with fulvic acid, reproduction was reduced significantly when compared with sodium humate or water treatments. Treatment of H. pseudorobustus with fulvic acid (pH 3.5) resulted in a greater reduction in reproduction in soil than did treatment with humic acid (pH 3.5).     

The pliocene species of Hipparion andtheir biostratigraphical meanings

During the Pliocene, the «Hipparion fauna in the Mediterranean area decreased sharply in comparison with the Upper Miocene. Nevertheless, this species can be used to represent the Hipparion biozones in order to compare them with the mammal units. H. gromovae characterizes the Ventian or MN 13 mammal unit; H. crassum the Ruscinian or the Lower Pliocene or MN 14 unit; H. fissurae the Upper Ruscinian or the Lower Pliocene, MN 15; H. rocinantis, the Middle Pliocene or Lower Villafranchian, MN 16a. This distribution can be correlated in most of the Eurasian region.     

Evaluation of the 23S rRNA gene as target for qPCR based quantification of Frankia in soils

The 23S rRNA gene was evaluated as target for the development of Sybr Green-based quantitative PCR (qPCR) for the analysis of nitrogen-fixing members of the genus Frankia or subgroups of these in soil. A qPCR with a primer combination targeting all nitrogen-fixing frankiae (clusters 1, 2 and 3) resulted in numbers similar to those obtained with a previously developed qPCR using nifH gene sequences, both with respect to introduced and indigenous Frankia populations. Primer combinations more specifically targeting three subgroups of the Alnus host infection group (cluster 1) or members of the Elaeagnus host infection group (cluster 3) were specific for introduced strains of the target group, with numbers corresponding to those obtained by quantification of nitrogen-fixing frankiae with both the 23S rRNA and nifH genes as target. Method verification on indigenous Frankia populations in soils, i.e. in depth profiles from four sites at an Alnus glutinosa stand, revealed declining numbers in the depth profiles, with similar abundance of all nitrogen-fixing frankiae independent of 23S rRNA or nifH gene targets, and corresponding numbers of one group of frankiae of the Alnus host infection only, with no detections of frankiae representing the Elaeagnus, Casuarina, or a second subgroup of the Alnus host infection groups.