Determination of genetic differences between fluid and nonfluid variants of<Emphasis Type="Italic">Clavibacter michiganensis</Emphasis> subsp.<Emphasis Type="Italic">sepedonicus</Emphasis> using rep-PCR technique

Testing of 23 isolates of Clavibacter michiganensis subsp. sepedonicus for analysis by rep-PCR (using BOX, ERIC, REP primer sets) was used for the purpose of localization of genetic markers for fluid and/or nonfluid strains. None of the primer sets was successful in detecting genetic differences between the isolates and no polymorphism was generated.     

Enhanced <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> immune responses caused by heterologous plant genes from <Emphasis Type="Italic">Pinellia ternata</Emphasis>

Background

Pinellia ternata is a Chinese traditional medicinal herb, used to cure diseases including insomnia, eclampsia and cervical carcinoma, for hundreds of years. Non-self-recognition in multicellular organisms can initiate the innate immunity to avoid the invasion of pathogens. A design for pathogen independent, heterosis based, fresh resistance can be generated in F₁ hybrid was proposed.

Results

By library functional screening, we found that P. ternata genes, named as ptHR375 and ptHR941, were identified with the potential to trigger a hypersensitive response in Nicotiana benthamiana. Significant induction of ROS and Callose deposition in N. benthamiana leaves along with activation of pathogenesis-related genes viz.; PR-1a, PR-5, PDF1.2, NPR1, PAL, RBOHB and ERF1 and antioxidant enzymes was observed. After transformation into N. benthamiana, expression of pathogenesis related genes was significantly up-regulated to generate high level of resistance against Phytophthora capsici without affecting the normal seed germination and morphological characters of the transformed N. benthamiana. UPLC-QTOF-MS analysis of ptHR375 transformed N. benthamiana revealed the induction of Oxytetracycline, Cuelure, Allantoin, Diethylstilbestrol and 1,2-Benzisothiazol-3(2H)-one as bioactive compounds. Here we also proved that F₁ hybrids, produced by crossing of the ptHR375 and ptHR941 transformed and non-transformed N. benthamiana, show significant high levels of PR-gene expressions and pathogen resistance.

Conclusions

Heterologous plant genes can activate disease resistance in another plant species and furthermore, by generating F₁ hybrids, fresh pathogen independent plant immunity can be obtained. It is also concluded that ptHR375 and ptHR941 play their role in SA and JA/ET defense pathways to activate the resistance against invading pathogens.

     

Comparative analysis of immune responses in Colorado potato beetle larvae during development of mycoses caused by <Emphasis Type="Italic">Metarhizium robertsii</Emphasis>, <Emphasis Type="Italic">M. brunneum</Emphasis>, and <Emphasis Type="Italic">M. pemphigi</Emphasis>

Development of mycoses and progress of humoral and cellular immune responses were compared in larvae of the Colorado potato beetle Leptinotarsa decemlineata infected with entomopathogenic fungi Metarhizium robertsii, M. brunneum and M. pemphigi. The larvae were found to be highly susceptible to the strains of M. robertsii and M. brunneum but weakly responsive to M. pemphigi. The extent of susceptibility to the pathogens was not related to the stimulating effect of epicuticular extracts on fungal growth. Metarhizium pemphigi, which is non-specific to the Colorado potato beetle, did not cause any significant changes in the immune response and did not colonize the hemocoel. When infected with M. robertsii and M. brunneum, the larvae exhibited an increase in hemocyte count during the early stage of mycosis (day 2) followed by a drastic decrease on day 3. The immunocompetent cells, plasmatocytes and granulocytes, exhibited the greatest decrease. Elevated phenoloxidase activity was recorded in the hemolymph and cuticle on days 2 and 3 post-infection. These changes in the immune responses correlated with strain-specific virulence. Thus, the immune response in Colorado potato beetle larvae is an important factor, which determines differences in the development of mycoses caused by different Metarhizium species.     

Genetic characterization and mapping of major gene resistance to potato leafroll virus in <Emphasis Type="Italic">Solanum</Emphasis><Emphasis Type="Italic">tuberosum</Emphasis> ssp. <Emphasis Type="Italic">andigena</Emphasis>

Major gene inheritance of resistance to Potato leafroll virus (PLRV) was demonstrated in a parthenogenic population derived from the highly resistant tetraploid andigena landrace, LOP-868. This major gene or chromosome region seems to control a single mechanism for resistance to infection and virus accumulation in this source. About 149 dihaploid lines segregated in a ratio of 107 resistant to 32 susceptible, fitting the expected ratio for inheritance of a duplex gene under random chromatid segregation. A tetraploid AFLP map was constructed using as reference the ultra high density (UHD) map. All AFLP markers associated with PLRV resistance mapped to the same linkage group. Map position was confirmed by analysis of previously-mapped SSR markers. Rl _adg is located on the upper arm of chromosome V, at 1 cM from its most closely linked AFLP marker, E35M48.192. This marker will be used to develop allele-specific primers or a pair of flanking PCR-based markers for their use in marker assisted selection.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration of <Emphasis Type="Italic">Alnus acuminata</Emphasis> H.B.K. ssp. <Emphasis Type="Italic">acuminata</Emphasis> and its root nodulation by <Emphasis Type="Italic">Frankia</Emphasis>

A procedure for in vitro plant regeneration of Alnus acuminata from epicotyls with cotyledonary buds was developed using different media formulations with different growth regulators and carbon sources. The development of multiple buds on explants at the initiation step was obtained with MS at 1/2 strength with either 1 or 2M of BAP but not without it. Multiplication gave up to 15 elongating shoots by explant, the best medium being MS supplemented with vitamins from B5 medium, 1M of BAP and 87mM sucrose. Rooting of about 88% occurred in the medium MS with 83 mM sucrose and 1M IBA. Alnus acuminata did not developed well on WPM. Roots of in vitro propagated plants were nodulated by Alnus-infective Frankia. The root nodules show a typical alder root nodule anatomy and differentiation pattern and effectively fixed nitrogen. Rhamnaceae-infective Frankia did not nodulate in vitro cultivated Alnus acuminata suggesting that symbiotic recognition was not altered by in vitro regeneration of the plant.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Synergistic action of entomopathogenic hyphomycetes and the bacteria <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> ssp. <Emphasis Type="Italic">morrisoni</Emphasis> in the infection of Colorado potato beetle <Emphasis Type="Italic">Leptinotarsa decemlineata</Emphasis>

A synchronous coinfection of the Colorado potato beetle Leptinotarsa decemlineata (Say) with the entomopathogenic bacteria Bacillus thuringiensis ssp. morrisoni Bonnifoi & de Barjak var. tenebrionis Krieg et al. and hyphomycete Metarhizium anisopliae (Metsch.) Sorokin or Beauveria bassiana (Bals.) Vuill leads to the rapid death of 95–100% of larvae. The bacteria arrest the nutrition of insects, while the fungal spores kill the weakened larvae. The synergistic effect of two pathogens is recorded at a relatively low hyphomycete titer (1–5 × 10⁶ conidia/ml) and is evident in the mortality dynamics at all larval ages. These bacterial and fungal pathogens display no antagonism on artificial nutrient media. This microbial complex is highly efficient under natural conditions (80–90% larval mortality rate and no plant defoliation).     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Ectomycorrhizae of <Emphasis Type="Italic">Tuber huidongense</Emphasis> and <Emphasis Type="Italic">T. liyuanum</Emphasis> with <Emphasis Type="Italic">Castanea mollissima</Emphasis> and <Emphasis Type="Italic">Pinus armandii</Emphasis>

Tuber huidongense and T. liyuanum are common commercial white truffles in China that belong to the Rufum and Puberulum groups of the genus Tuber, respectively. Their mycorrhizae were successfully synthesized with two native trees—Castanea mollissima and Pinus armandii—under greenhouse conditions. The identities of the mycorrhizae were confirmed through internal transcribed spacer (ITS) sequence analyses, and their morphological characteristics were described. All of the obtained mycorrhizae have an interlocking pseudoparenchymatous mantle, which is a typical feature of truffle mycorrhizae. The mycorrhizae of T. huidongense on the two trees have hyaline branched emanating hyphae, similar to the documented mycorrhizae of the Rufum group. The unramified, spiky, and hyaline cystidia on the mycorrhizae of T. liyuanum with both C. mollissima and P. armandii further confirmed that this characteristic is constant for the mycorrhizae of the Puberulum group. The successful mycorrhizal syntheses on the two nut-producing trees will be of economic importance in the cultivation of the two truffles.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

Activation of stilbene synthesis in cell cultures of <Emphasis Type="Italic">Vitis amurensis</Emphasis> by calcium-dependent protein kinases <Emphasis Type="Italic">VaCPK1</Emphasis> and <Emphasis Type="Italic">VaCPK26</Emphasis>

Stilbenes, including trans-resveratrol (3,4′,5-trihydroxy-trans-stilbene), are known to exert beneficial health effects and contribute to plant biotic stress resistance. Much remains to be discovered about the cell signaling pathways regulating stilbene biosynthesis. It has recently been shown that overexpression of the calcium-dependent protein kinase VaCPK20 gene considerably increased t-resveratrol accumulation in cell cultures of Vitis amurensis. In this study, we analyzed the involvement of other CDPK family members, VaCPK1 and VaCPK26, on stilbene synthesis and biomass production by cell cultures of V. amurensis. We showed that overexpression of the VaCPK1 and 26 genes induced production of stilbenes by 1.7–4.6-fold (for VaCPK1) and by 2.5–6.2-fold (for VaCPK26) in several independently established cell lines compared to the empty vector-transformed control. Using HPLC-UV-MS, we detected five stilbenes in the grape cells: t-resveratrol diglucoside, t-piceid, t-resveratrol, ε- and δ-viniferin. The VaCPK1- and VaCPK26-transformed calli were capable of producing 1.4–3.1 and 1.8–4.9 mg/l of t-resveratrol, respectively (up to 0.4 for and 0.6 mg/g of dry weight for VaCPK26 and VaCPK1, respectively), while the control line synthesized only 0.5 mg/l of t-resveratrol (0.07 mg/g DW). The up-regulation of t-resveratrol production in the VaCPK1- and VaCPK26-overexpressing grape calli correlated with a significant up-regulation of stilbene synthase (STS) gene expression, especially VaSTS7. The data indicate that VaCPK1 and 26 genes, which are close homologues of VaCPK20, are positive regulators of stilbene biosynthesis in grapevine.     

Species identification of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> ssp. <Emphasis Type="Italic">jejuni</Emphasis> and <Emphasis Type="Italic">C. coli</Emphasis> by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and PCR

The Campylobacter species strains (n = 42; isolated from clinical samples and deposited in Czech National Collection of Type Cultures, Prague) originally phenotypically (and biochemically) identified as Campylobacter jejuni were re-classified using molecular biological and mass spectrometric methods. Whole-cell MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) separated the isolates into two genetically related strains — C. jejuni (n = 26) and C. coli (n = 16) and, moreover, distinguished the intimate details in the group of tested strains. It also made it possible to create the MALDI-TOF MS dendrogram; similarly, the spectral characteristics were used for the 3D cluster analysis. Polymerase chain reaction (PCR) confirmed the results obtained by mass spectrometry. Both methods (PCR and MALDI-TOF MS) gave the same results which supports their suitability in the rapid and accurate Campylobacter-species determination. Part of this work was presented at the 24th Congress of Czechoslovak Society for Microbiology, Liberec (Czech Republic) 2007.