Endophytic fungi assemblages from 10 <Emphasis Type="Italic">Dendrobium</Emphasis> medicinal plants (Orchidaceae)

Dendrobium is the largest genus of tropical epiphytic orchid, some of which are traditional Chinese medicinal plants. The therapeutic components varied significantly among species. Endophytic microbes (fungi) hidden in medicinal plants may play an important effect on the overall quality of herb. Investigation of fungal composition in host plants is the first step toward elucidating the relationship endophyte-therapeutic content of herbal medicine. In this study, 401 culturable fungal endophytes were isolated and identified from 10 species of medicinal Dendrobium based on morphological and molecular techniques. The results showed that endophytic fungi from Dendrobium plants exhibited high biodiversity (37 genera, about 80 species). Acremonium, Alternaria, Ampelomyces, Bionectria, Cladosporium, Colletotrichum, Fusarium, Verticillium and Xylaria were the dominant fungal endophytes. Tropical epiphytic orchids appear to vary in degree of host specificity in their endophytic fungi.     

In situ seed baiting techniques in <Emphasis Type="Italic">Dendrobium officinale</Emphasis> Kimuraet Migo and <Emphasis Type="Italic">Dendrobium nobile</Emphasis> Lindl. : the endangered Chinese endemic <Emphasis Type="Italic">Dendrobium</Emphasis> (Orchidaceae)

The new in situ seed baiting method using seed packets to assess germination of orchid species within soil provides a means of locating, collecting, and identifying specific fungi that are involved in the lifecycle of orchids in the wild and investigating their relationship with the orchids under natural field conditions. Two isolates (SHH44, SHH53) originating from the seedlings found in the seeds packets in situ were demonstrated to support seed germination and seedling development (in vitro) of two endangered Chinese endemic herbs, Dendrobium officinale and Dendrobium nobile (Orchidaceae). Advanced protocorm development (Stage 3 and greater) in this study of these species of Dendrobium only occurred under the 12/12 h L/D photo-period indicated that illumination may play an important role in seedling recruitment of terrestrial orchid species in their natural environment. The information provided in this study may prove invaluable in the conservation of D. officinale and D. nobile and congener species in China, especially given their rare status.     

Methods for genetic transformation in <Emphasis Type="Italic">Dendrobium</Emphasis>

Key message

The genetic transformation of Dendrobium orchids will allow for the introduction of novel colours, altered architecture and valuable traits such as abiotic and biotic stress tolerance.

Abstract

The orchid genus Dendrobium contains species that have both ornamental value and medicinal importance. There is thus interest in producing cultivars that have increased resistance to pests, novel horticultural characteristics such as novel flower colours, improved productivity, longer flower spikes, or longer post-harvest shelf-life. Tissue culture is used to establish clonal plants while in vitro flowering allows for the production of flowers or floral parts within a sterile environment, expanding the selection of explants that can be used for tissue culture or genetic transformation. The latter is potentially the most effective, rapid and practical way to introduce new agronomic traits into Dendrobium. Most (69.4 %) Dendrobium genetic transformation studies have used particle bombardment (biolistics) while 64 % have employed some form of Agrobacterium-mediated transformation. A singe study has explored ovary injection, but no studies exist on floral dip transformation. While most of these studies have involved the use of selector or reporter genes, there are now a handful of studies that have introduced genes for horticulturally important traits.

     

<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.     

Induction of <Emphasis Type="Italic">in vitro</Emphasis> flowering in the orchid <Emphasis Type="Italic">Dendrobium</Emphasis> Sonia 17

In this study, Dendrobium Sonia 17 plantlets were used to induce in vitro flowering. Inflorescences were induced and rooting was inhibited in the half-strength Murashige and Skoog medium containing 20 μM N ⁶-benzyladenine (BA). The medium with high P and low N contents was effective to induce inflorescences while the medium with low P and high N contents was only effective to promote forming of shoots. In addition, the induced in vitro inflorescences were able to multiply and maintain without exhibiting a distinctive vegetative phase. Different morphologies of in vitro flowers such as incomplete flower structures, abnormal and unresupinated in vitro flowers were observed.     

Endophytic <Emphasis Type="Italic">Penicillium citrinum</Emphasis> Thom. from <Emphasis Type="Italic">Scoparia dulcis</Emphasis> Linn

Scoparia dulcis of Scrophulariaceae is an annual herb distributed through out the tropics. Penicillium citrinum was obtained from apparently healthy roots, stem, leaves and fruits of this plant. Callus and multiple shoots produced during micropropagation from various explants were also symptomless but showed occurrence of Penicillium citrinum when cultured in Murashige & Skoog liquid medium for the production of secondary metabolites.     

Endophytic fungi from rhizomes of <Emphasis Type="Italic">Paris polyphylla</Emphasis> var. <Emphasis Type="Italic">yunnanensis</Emphasis>

About 63 fungal endophytic isolates were separated from rhizomes of Paris polyphylla var. yunnanensis, which is a traditional medicinal plant mainly distributed in China. The isolates were characterized and grouped based on the culture characteristics and the morphology of colony growth and conidia. Eleven representative ones were selected for further taxonomical identification. Five genera namely Fusarium, Gliocladiopsis, Gliomastix, Aspergillus and Cylindrocarpon were identified on the basis of their morphological characterizations. Of them, the most frequent genus was Fusarium (i.e. Ppf1, Ppf3 and Ppf14). Their ITS-rDNA sequences were compared with those available in the GeneBank databases to obtain the closest related species by BLAST analysis as well as to analyze their phylogenetic affiliation. The isolates were identified as Gliocladiopsis irregularis (Ppf2), Plectosphaerella cucumerina (Ppf4), Padospora sp. (Ppf6), Gliomastix murorum var. murorum (Ppf7), Aspergillus fumigatus (Ppf9), Pichia guilliermondii (Ppf10), Neonectria radicicola (anamorph: Cylindrocarpon) (Ppf12) and one uncultured mycorrhizal ascomycete (Ppf13) separately based on their morphological and molecular features. The molecular characters of the endophytic fungi were basically coincident with their morphology. The broad diversity and taxonomic spectrum were exhibited by the endophytic fungi from P. polyphylla var. yunnanensis.     

<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.     

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.     

<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.     

Diversity and recombination in <Emphasis Type="Italic">Wolbachia</Emphasis> and <Emphasis Type="Italic">Cardinium</Emphasis> from <Emphasis Type="Italic">Bryobia</Emphasis>spider mites

BACKGROUND: Wolbachia and Cardinium are endosymbiotic bacteria infecting many arthropods and manipulating host reproduction. Although these bacteria are maternally transmitted, incongruencies between phylogenies of host and parasite suggest an additional role for occasional horizontal transmission. Consistent with this view is the strong evidence for recombination in Wolbachia, although it is less clear to what extent recombination drives diversification within single host species and genera. Furthermore, little is known concerning the population structures of other insect endosymbionts which co-infect with Wolbachia, such as Cardinium. Here, we explore Wolbachia and Cardinium strain diversity within nine spider mite species (Tetranychidae) from 38 populations, and quantify the contribution of recombination compared to point mutation in generating Wolbachia diversity. RESULTS: We found a high level of genetic diversity for Wolbachia, with 36 unique strains detected (64 investigated mite individuals). Sequence data from four Wolbachia genes suggest that new alleles are 7.5 to 11 times more likely to be generated by recombination than point mutation. Consistent with previous reports on more diverse host samples, our data did not reveal evidence for co-evolution of Wolbachia with its host. Cardinium was less frequently found in the mites, but also showed a high level of diversity, with eight unique strains detected in 15 individuals on the basis of only two genes. A lack of congruence among host and Cardinium phylogenies was observed. CONCLUSIONS: We found a high rate of recombination for Wolbachia strains obtained from host species of the spider mite family Tetranychidae, comparable to rates found for horizontally transmitted bacteria. This suggests frequent horizontal transmission of Wolbachia and/or frequent horizontal transfer of single genes. Our findings strengthens earlier reports of recombination for Wolbachia, and shows that high recombination rates are also present on strains from a restrictive host range. Cardinium was found co-infecting several spider mite species, and phylogenetic comparisons suggest also horizontal transmission of Cardinium among hosts.     

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.     

<Emphasis Type="Italic">Diplazium</Emphasis> hybrids involving <Emphasis Type="Italic">D. plantaginifolium</Emphasis> and <Emphasis Type="Italic">D</Emphasis>. <Emphasis Type="Italic">ternatum</Emphasis> from Mexico and Central America

Here we evaluate the origins and relationships of Mexican and Central American Diplazium hybrids derived from crosses involving either D. plantaginifolium or D. ternatum. Based on study of live plants and herbarium specimens, we distinguish D. ×verapax from the similar D. riedelianum and present evidence that the former is a sterile hybrid derived from a cross between D. plantaginifolium and D. werckleanum. We also describe new hybrids, D. ×torresianum and D. ×subternatum from Mexico and northern Central America. Both involve D. ternatum as one parent. Diplazium. cristatum is the other putative parent of D. ×torresianum, and D. plantaginifolium is the second parent of D. ×subternatum. We also designate lectotypes for D. cordovense and D. dissimile.     

Endophytic <Emphasis Type="Italic">Alcaligenes</Emphasis> Isolated from Horticultural and Medicinal Crops Promotes Growth in Okra (<Emphasis Type="Italic">Abelmoschus</Emphasis><Emphasis Type="Italic">esculentus</Emphasis>)

The potential of endophytic bacteria to act as biofertilizers and bioprotectants has been demonstrated, and considerable progress has been made in explaining their role in plant protection. In the present study, three endophytic bacterial strains (BHU 12, BHU 16 isolated from the leaves of Abelmoschus esculentus, and BHU M7 isolated from the leaves of Andrographis paniculata) were used which displayed high sequence similarity to Alcaligenes faecalis. The biofilm formation ability of these endophytic strains in the presence of okra root exudates confirms their chemotactic ability, an initial step for successful endophytic colonization. Further, reinoculation of spontaneous rifampicin-tagged mutants into okra seedlings revealed a CFU count above 10⁵ cells g^?1 of all three endophytic strains in root samples during the first 15 days of plant growth. The CFU count increased up to 10¹³ by 30 days of plant growth, followed by a gradual decline to approximately 10¹⁰ cells g^?1 at 45 days of plant growth. Systemic endophytic colonization was further supported by 2, 3, 5-triphenyl tetrazolium chloride staining and fluorescence imaging of ds-RED expressing conjugants of the endophytic strains. The strains were further assessed for their plausible in vivo and in vitro plant growth-promoting and antagonistic abilities. Our results demonstrated that the endophytic strains BHU 12, BHU 16, and BHU M7 augmented plant biomass by greater than 40 %. Root and shoot lengths of okra plants when primed by BHU 12, BHU 16, and BHU M7 increased up to 34 and 14.5 %, respectively. The endophytic isolates also exhibited significant in vitro antagonistic potential against the collar rot pathogen Sclerotium rolfsii. In summary, our results demonstrate excellent potential of the three endophytic bacterial strains as biofertilizers and biocontrol agents, indicating the possibility for use in sustainable agriculture.     

Identification,characterization, and expression of the <Emphasis Type="Italic">SWEET</Emphasis> gene family in <Emphasis Type="Italic">Phalaenopsis equestris</Emphasis> and <Emphasis Type="Italic">Dendrobium officinale</Emphasis>

Sugars are important molecules that function not only as primary metabolites, but also as nutrients and signal molecules in plants. The sugar transport protein genes family SWEET has been recently identified. The availability of the Dendrobium officinale and Phalaenopsis equestris genome sequences offered the opportunity to study the SWEET gene family in this two orchid species. We identified 22 and 16 putative SWEET genes, respectively, in the genomes of D. officinale and P. equestris using comprehensive bioinformatics analysis. Based on phylogenetic comparisons with SWEET proteins from Arabidopsis and rice, the DoSWEET and PeSWEET proteins could be divided into four clades; among these, clade II specifically lacked PeSWEETs and clade IV specifically lacked DoSWEETs, and there were orthologs present between D. officinale and P. equestris. Protein sequence alignments suggest that there is a predicted serine phosphorylation site in each of the highly conserved MtN3/saliva domain regions. Gene expression analysis in four tissues showed that three PeSWEET genes were most highly expressed in the flower, leaf, stem, and root, suggesting that these genes might play important roles in growth and development in P. equestris. Analysis of gene expression in different floral organs showed that five PeSWEET genes were highly expressed in the column (gynostemium), implying their possible involvement in reproductive development in this species. The expression patterns of seven PeSWEETs in response to different abiotic stresses showed that three genes were upregulated significantly in response to high temperature and two genes were differently expressed at low temperature. The results of this study lay the foundation for further functional analysis of SWEET genes in orchids.     

Prevalence,intensity and associated factor analysis of <Emphasis Type="Italic">Tropilaelaps</Emphasis><Emphasis Type="Italic">mercedesae</Emphasis> infesting <Emphasis Type="Italic">Apis</Emphasis><Emphasis Type="Italic">mellifera</Emphasis> in China

Tropilaelaps mercedesae is a serious ectoparasite of Apis mellifera in China. The aim of this study was to investigate the infestation rates and intensity of T. mercedesae in A. mellifera in China, and to explore the relative importance of climate, district, management practices and beekeeper characteristics that are assumed to be associated with the intensity of T. mercedesae. Of the 410 participating apiaries, 379 apiaries were included in analyses of seasonal infestation rates and 352 apiaries were included in multivariable regression analysis. The highest infestation rate (86.3%) of T. mercedesae was encountered in autumn, followed by summer (66.5%), spring (17.2%) and winter (14.8%). In autumn, 28.9% (93) of the infested apiaries were in the north (including the northeast and northwest of China), 71.1% (229) were in the central and south (including east, southeast and southwest China), and 306 apiaries (82.9%) were co-infested by both T. mercedesae and Varroa. Multivariable regression analysis showed that geographical location, season, royal jelly collection and Varroa infestation were the factors that influence the intensity of T. mercedesae. The influence of beekeeper’s education, time of beekeeping, operation size, and hive migration on the intensity of T. mercedesa was not statistically significant. This study provided information about the establishment of the linkage of the environment and the parasite and could lead to better timing and methods of control.