Illumina-based analysis of the rhizosphere microbial communities associated with healthy and wilted Lanzhou lily (<Emphasis Type="Italic">Lilium davidii</Emphasis> var. <Emphasis Type="Italic">unicolor</Emphasis>) plants grown in the field

Lanzhou lily (Liliumdavidii var. unicolor) is the best edible lily as well as a traditional medicinal plant in China. The microbes associated with plant roots play crucial roles in plant growth and health. However, little is known about the differences of rhizosphere microbes between healthy and wilted Lanzhou lily (Lilium davidii var. unicolor) plants. The objective of this study was to compare the rhizosphere microbial community and functional diversity of healthy and wilted plants, and to identify potential biocontrol agents with significant effect. Paired end Illumina Mi-Seq sequencing of 16S rRNA and ITS gene amplicons was employed to study the bacterial and fungal communities in the rhizosphere soil of Lanzhou lily plants. BIOLOG technology was adopted to investigate the microbial functional diversity. Our results indicated that there were major differences in the rhizosphere microbial composition and functional diversity of wilted samples compared with healthy samples. Healthy Lanzhou lily plants exhibited lower rhizosphere-associated bacterial diversity than diseased plants, whereas fungi exhibited the opposite trend. The dominant phyla in both the healthy and wilted samples were Proteobacteria and Ascomycota, i.e., 34.45 and 64.01 %, respectively. The microbial functional diversity was suppressed in wilted soil samples. Besides Fusarium, the higher relative abundances of Rhizoctonia, Verticillium, Penicillium, and Ilyonectria (Neonectria) in the wilted samples suggest they may pathogenetic root rot fungi. The high relative abundances of Bacillus in Firmicutes in healthy samples may have significant roles as biological control agents against soilborne pathogens. This is the first study to find evidence of major differences between the microbial communities in the rhizospheric soil of healthy and wilted Lanzhou lily, which may be linked to the health status of plants.     

Investigation of karyotypic composition and evolution in <Emphasis Type="Italic">Lilium</Emphasis> species belonging to the section martagon

Analyzing chromosomal traits is one of the pragmatic ways to establish evolutionary and genetic database of plants that has complicated phylogenetic system. There are some conflicts on the exact phylogeny and evolutionary pathway of Lilium, and section martagon is the most complicated part among them. In this study, chromosomal traits of martagon lily species are described. All martagon lilies were analyzed with FISH (Fluorescence in situ hybridization) technique, followed by detailed karyotyping. Each species showed 2n = 2x = 24 of chromosome complement. Size of chromosomes ranged from 451.04 to 680.06 µm. 5S and 45S ribosomal DNA, general molecular markers in modern evolutionary research were used as probe in this study. Variation in rDNA loci and chromosome translocation were observed in Lilium hansonii; the highest number of 45S rDNA loci was detected in Lilium hansonii, followed by other martagon lilies, in similar locations but with differences, and chromosome translocation was observed from one individual of Lilium hansonii. Additionally, Lilium tsingtauense from Jeju-do Island, Korea was detected with two extra chromosomes. These kind of genetic variations through karyotyping indicate ongoing genetic variations in martagon lilies. In this study, precise analysis of chromosome traits in Lilium species belonging to section martagonperformed to contribute to better comprehension of the evolutionary pathway and establishment of cytogenetic database for further plant breeding research.     

The use of miRNAs as reference genes for miRNA expression normalization during <Emphasis Type="Italic">Lilium</Emphasis> somatic embryogenesis by real-time reverse transcription PCR analysis

Expression profiling of miRNAs has the ability to reveal the essence of somatic embryogenesis (SE). qRT-PCR is one of the most commonly used techniques for dynamic miRNA detection but requires optimal reference genes for data reliability. This is the first report on reference gene validation for miRNA expression normalization in Lilium (Lilium pumilum DC. Fisch. and Lilium davidii var. unicolor). In this study, seventeen miRNAs together with two snRNAs (U4, U6), one rRNA (5S rRNA) and three protein-coding genes (FP, ACT, GAPDH) were selected as reference candidates, and their expression stability was validated by qRT-PCR among eleven developing SE cultures in two lilies. Four normalization algorithms, including geNorm, BestKeeper, NormFinder and RefFinder, were also used to evaluate the stability of the reference candidates. For Lilium pumilum DC. Fisch., lpu-miR159a was the optimal reference gene during SE, followed by lpu-miR408b, while U6 was the least stable reference candidate. For Lilium davidii var. unicolor, FP presented greater stability than did half of the miRNA candidates, but the best reference gene was lda-miR162, followed by lda-miR159a. Further analysis of the expression level of miR156 and miR529 was used to evaluate the validity of the reference genes in both lilies. In general, miRNAs are superior to common protein-coding genes and snRNAs / rRNAs as reference genes for miRNA expression normalization during Lilium SE, and the most suitable reference miRNA is different between two species in the same Lilium genus. This is a pioneer study using suitable miRNAs as reference genes in Lilium and constitutes a small but essential step for the further exploration of miRNA function in Lilium, thus offering valuable references for other plants.     

Micropropagation of Ajuga species: a mini review

The genus Ajuga L., belonging to Lamiaceae family, is widespread. The demand for Ajuga species has risen sharply because of their medicinal, ornamental, and pharmacological properties. These wide-ranging plants are being rapidly depleted due to over-collection for ornamental and medicinal purposes, as well as by habitat destruction and deforestation. Ajuga boninsimae, A. bracteosa, A. ciliate, A. genevensis, A. incisa, A. makinoi, A. multiflora, A. pyramidalis, A. shikotanensis, A. reptans, and A. vestita are categorized and protected as endangered plants. In vitro plant culture has therefore emerged for the conservation and mass clonal propagation of rare plants. This mini-review covers the current in vitro scenario in the propagation of Ajuga species. Adventitious or axillary shoots are initiated on the leaf, petiole and internodes, as well as roots, nodes, and shoot tip explants. Shoot induction is predominantly dependent on plant growth regulators added to the culture medium. Full- or half-strength Murashige and Skoog medium with or without auxin is used for in vitro rooting. Rooted shoots need to be acclimatized in the greenhouse with an estimated 82–100% survival rate.     

Effects of endogenous hormones on variation of shoot branching in a variety of non-heading Chinese cabbage and related gene expression

Shoot branching (tillering) primarily determines plant shoot architecture and has been studied in many plants. Shoot branching is an important trait in non-heading Chinese cabbage (Brassica rapa ssp. chinensis Makino). The B. rapa ssp. chinensis var. multiceps exhibits unique and multiple shoot branching characteristics. Here, we analyzed the variation in shoot branching between ‘Maertou,’ with multiple shoot branching, and ‘Suzhouqing,’ a common variety. The levels of endogenous indole-3-acetic acid (IAA), zeatin riboside and active gibberellins in the shoot meristem tissues of the two cultivars were quantified by enzyme-linked immunosorbent assay during the vegetative growth stage. High levels of IAA maintained axillary bud dormancy and repressed axillary bud outgrowth allowing shoot branching to form in the vegetative stage in ‘Suzhouqing.’ In contrast, low levels of IAA did not inhibit axillary buds in ‘Maertou,’ while a high level of cytokinin promoted axillary bud growth and branch shoot development. Exogenous hormone (rac-GR24 and 6-benzylaminopurine) treatment showed that ‘Maertou’ was relatively sensitive to cytokinin, because the fold changes of cytokinin-responsive genes in ‘Maertou’ were significantly more frequent than those in ‘Suzhouqing’. Cytokinin was the direct regulator for axillary bud growth of ‘Maertou’. Compared with ‘Suzhouqing’, ‘Maertou’ was sensitive to cytokinin and this weakened the strigolactone–cytokinin branching pathway.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of endangered <Emphasis Type="Italic">Mammillaria</Emphasis> genus (Cactaceae) species and genetic stability assessment using SSR markers

In vitro propagation protocols were established for endangered species of cacti Mammillaria hernandezii, M. dixanthocentron, and M. lanata. In vitro-germinated seedlings were used as the explant source. Three explant types were evaluated as apical, basal, and lateral stem sections. Shoot multiplication was achieved using Murashige and Skoog (MS) medium supplemented with benzyladenine, kinetin, meta-topolin, and thidiazuron in equimolar concentrations (0.0, 0.4, 1.1, 2.2, 4.4, and 8.9 μM). Shoot regeneration was obtained primarily in the lateral stem section explants. In M. hernandezii, an average of 7.4 shoots was regenerated in MS medium with 2.2 μM meta-topolin. M. dixanthocentron and M. lanata averaged 16.7 and 17.9 shoots/explant, respectively, in MS medium supplemented with 1.1 μM meta-topolin. Rooting occurred in MS medium without growth regulators. Three in vitro culture cycles were performed to validate the propagation protocols and to verify genetic stability. Shoots were collected in each cycle and genomic DNA was extracted. Amplified microsatellites were used to compare each genotype with its respective donor plant. Polymorphic information content analysis showed low levels of intra-clonal polymorphisms—M. hernandezii 0.04 and M. dixanthocentron and M. lanata both 0.12. More than 95% of the plants were successfully acclimatized in the greenhouse. After 12 months, plants of M. hernandezii reached the flowering stage; M. dixanthocentron and M. lanata flowered at 24 mo.     

A novel subspecies of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter africanus’ found on native <Emphasis Type="Italic">Teclea gerrardii</Emphasis> (Family: Rutaceae) from South Africa

The phloem limited bacterium ‘Candidatus Liberibacter africanus’ is associated with citrus greening disease in South Africa. This bacterium has been identified solely from commercial citrus in Africa and the Mascarene islands, and its origin may lie within an indigenous rutaceous host from Africa. Recently, in determining whether alternative hosts of Laf exist amongst the indigenous rutaceous hosts of its triozid vector, Trioza erytreae, three novel subspecies of Laf were identified i.e. ‘Candidatus Liberibacter africanus subsp. clausenae’, ‘Candidatus Liberibacter africanus subsp. vepridis’ and ‘Candidatus Liberibacter africanus subsp. zanthoxyli’ in addition to the formerly identified ‘Candidatus Liberibacter africanus subsp. capensis’. The current study expands upon the range of indigenous rutaceous tree species tested for liberibacters closely related to Laf and its subspecies. A collection of 121 samples of Teclea and Oricia species were sampled from Oribi Gorge and Umtamvunu nature reserves in KwaZulu Natal. Total DNA was extracted and the presence of liberibacters from these samples determined using a generic liberibacter TaqMan real-time PCR assay. Liberibacters from positive samples were further characterised through amplification and sequencing of the 16S rRNA, outer-membrane protein (omp) and 50S ribosomal protein L10 (rplJ) genes. A single Teclea gerrardii specimen tested positive for a liberibacter and, through phylogenetic analyses of the three genes sequenced, was shown to be unique, albeit closely related to ‘Ca. L. africanus’ and ‘Ca. L. africanus subsp. zanthoxyli’. We propose that this newly identified liberibacter be named ‘Candidatus Liberibacter africanus subsp. tecleae’.     

Effects of virus infection on plant growth,root development and phytohormone levels in in vitro-cultured pear plants

Virus infection is common in pear (Pyrus spp.) trees commercially cultivated in China. In this study, the growth speed, root development and phytohormone levels of virus-free and virus-infected pear cultures were comparatively investigated. Results showed that the co-infection of Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV) significantly decreased the growth and proliferation of in vitro plants of P. communis cv. ‘Confenence’, but showed relatively less effect for P. pyrifolia cv. ‘Jinshui no. 2’, P. communis cv. ‘Red Clapp Favonite’ and P. sinkiangensis cv. ‘Korla’. Strong inhibition of ASGV infection to root development was observed for in vitro plants of ‘Jinshui no. 2’, but not for ‘Confenence’. Furthermore, during root induction, ASGV infection significantly increased CTKs/IAA ratio and decreased IAA/ABA ratio in the rooting region of in vitro plants of ‘Jinshui no. 2’. Nevertheless, for in vitro plants of ‘Confenence’, these values were rarely influenced by ASGV and ASPV infection. The result indicated that the changes of CTKs/IAA and IAA/ABA ratios might be responsible for rooting inhibition of in vitro plants of ‘Jinshui no. 2’.     

<Emphasis Type="Italic">Cytisus aeolicus</Emphasis> Guss. ex Lindl. <Emphasis Type="Italic">in vitro</Emphasis> cultures and genistin production

Cytisus aeolicus Guss. ex Lindl. (Fabaceae family, subfamily Faboideae) is an endangered endemic species of the Aeolian Islands, Sicily. In vitro multiplication of C. aeolicus shoots was described in this work and cell cultures were established from cotyledons and hypocotyls to investigate their potential production of isoflavones. Aseptically germinated seeds, cultivated on LS modified basal medium, gave the initial explants used both to induce axillary propagation and callus cultures. The LS (Linsmaier and Skoog) basal medium, supplemented with 0.1 mg L^?1 of 6-benzylaminopurine were used to induce axillary propagation. The callus induction was performed using the basal medium added with 5 mg L^?1 2,4-dichlorophenoxy acetic acid and 5 mg L^?1 kinetin (control medium). Basal medium was also added with 2000 mg L^?1 casein hydrolysate (CH) or 900 mg L^?1myo-inositol (MI). C. aeolicus callus cultures on CH and MI media produced an unique compound, the isoflavone genistein 7-O-ß-D-glucopyranoside (genistin), which has not previously been isolated from wild plants. Callus cultures grown on the medium containing myo-inositol produced the greatest amount of genistin. C. aeolicus tissue culture procedures could provide suitable plant material both for germplasm preservation (by micropropagation) and for biotechnological selective isoflavone production (by callus culture).     

New species of Telenominae (Hymenoptera,Scelionidae) from the palaearctic fauna

Four new species of Telenominae of the genera Telenomus and Platytelenomus, collected in the territory of the Ukraine, Hungary, and Japan, were described: Telenomus (T.) bicolorus Kononova, T. (T.) ardens Kononova, (T.) michaylovi Kononova, and Platytelenomus mirabilis Kononova. Brief morphological characteristics of the genera Telenomus and Platytelenomus are given, and some notes concerning biology and geographical distribution of the species are presented. Telenomus (T.) bicolorus differs from all the known species of the genus Telenomus in the two-colored body: head and thorax yellow with brownish tint ventrally, mesothorax and abdomen black. The main distinguishing feature of T. (T.) ardens is its smooth shining body, T. (T.) michaylovi is similar to T. (T.) rudis Kozlov. These species can be distinguished by the structure of their antenna. The second to fourth segments of the antennal clava are transverse in T. (T.) michaylovi and are as long as wide in T. (T.) rudis. In addition, the abdominal stem and abdominal tergite II are smooth and shining, while the abdominal stem in T. (T.) rudis is striate along the entire length, and tergite II is finely striate along half of its length. Platytelenomus mirabilis is closely related to P. danubialis Szelényi, but differs in the strongly flattened body, sculpture of the abdominal stem and tergite II, and coloration of the legs. The thorax of P. mirabilis is 4–5 times as wide as high, the abdominal stem is striate along the entire length, tergite II is striate at the base, and the legs, including coxae, are yellow. The thorax of P. danubialis is 4 times as wide as high, the abdominal stem and tergite II are smooth and shining, and the legs are brown.     

Nucleolar RNA Synthesis during Meiosis of Lily Microsporocytes

SEVERAL authors have reported a decrease in nucleolar incorporation of ³H-uridine into RNA in male gametocytes of maize, locusts and mammals during meiotic prophase^1–4, but the inactive nucleolus often persists. In the microsporocytes of Liliutn henryi the cytoplasmic ribosomes reportedly decrease in number during the extended meiotic prophase as the cellular RNA concentration also decreases⁵. Stern (personal communication) has also observed a decrease in RNA content in meiotic cells of Lilium longiflorum. We have examined the RNA synthetic activities of lily microsporocytes to see if the large nucleolus present is engaged in the synthesis of ribosomal RNA.     

Biotechnological advances in <Emphasis Type="Italic">Lilium</Emphasis>

Modern powerful techniques in plant biotechnology have been developed in lilies (Lilium spp., Liliaceae) to propagate, improve and make new phenotypes. Reliable in vitro culture methods are available to multiply lilies rapidly and shorten breeding programs. Lilium is also an ideal model plant to study in vitro pollination and embryo rescue methods. Although lilies are recalcitrant to genetic manipulation, superior genotypes are developed with improved flower colour and form, disease resistance and year round forcing ability. Different DNA molecular markers have been developed for rapid indirect selection, genetic diversity evaluation, mutation detection and construction of Lilium linkage map. Some disease resistance-QTLs are already mapped on the Lilium linkage map. This review presents latest information on in vitro propagation, genetic engineering and molecular advances made in lily.     

Susceptibility of germinating cruciferous seeds to <Emphasis Type="Italic">Bagrada hilaris</Emphasis> (Hemiptera: Pentatomidae) feeding injury

Bagrada hilaris (Burmeister) (Hemiptera: Pentatomidae) is a serious pest that attacks both germinating and seedling stages of a variety of cruciferous crops grown in the Central Coast of California. B. hilaris feeding on germinating seeds can cause severe stunting and plant mortality, and little is known about the feeding preference of B. hilaris for germinating seeds of major cruciferous hosts and varieties of hosts. No-choice and choice experiments were conducted in which germinating seeds in soilless and soil settings were exposed to B. hilaris adults for 7 days. Susceptibility scores were developed using B. hilaris feeding injury sites, distorted leaves, and deformed and dead plants to determine the overall B. hilaris preference for germinating host seeds. Based on the scores, the order of preference was arugula (Eruca sativa L.)?>?turnip (B. rapa L. var. rapa)?>?mizuna (B. rapa L. nipposinica)?>?kale (B. oleracea L. acephala)?>?choi (Brassica rapa L. var. chinensis)?>?broccoli (B. oleracea var. italica Plenck)?>?cauliflower (B. oleracea L. var. botrytis)?>?lettuce (Lactuca sativa L.)?>?sweet alyssum (Lobularia maritima [L.] Desv.). The lowest feeding injury was recorded on germinating lettuce and sweet alyssum seeds. Furthermore, no-choice and choice experiments were conducted with four varieties each of arugula and mizuna, twelve varieties each of kale and choi, and nine varieties/types of leafy Asian greens. The arugula varieties ‘Wild Rocket’ and ‘Spirit’ were more damaged by B. hilaris than other varieties tested. Among mizuna varieties, ‘Beira F1’ was more attractive to B. hilaris than ‘Scarlet’ or ‘Starbor F1.’ The choi varieties ‘Tokyo Bekana,’ ‘Feng Qing Choi F1,’ ‘Joi Choi F1,’ and ‘Win-Win Choi F1’ were more attractive than ‘Rosie F1.’ The leafy Asian greens variety ‘Carlton F1’ was more attractive to B. hilaris than ‘Yukina Savon,’ ‘Tatsoi OG,’ ‘Komatsuna Summerfest F1,’ ‘Red Rain F1,’ and ‘Shungiku.’ Therefore, the results suggest that not all varieties were equally susceptible to B. hilaris feeding and possibly be utilized for further field evaluation as a trap crop or developing more resistant varieties to B. hilaris.     

New phylogenetic insights into <Emphasis Type="Italic">Saprolegniales</Emphasis> (Oomycota,Straminipila) based upon studies of specimens isolated from Brazil and Argentina

Saprolegniales is a complex and monophyletic order of oomycetes. Their members inhabit terrestrial, freshwater, and marine ecosystems and have a worldwide distribution. In these ecosystems, they are found as saprobes, parasites, or even pathogens of animals and plants of economic importance. In this study, a concatenate phylogeny of the partial LSU and complete ITS rDNA regions is presented, including isolates from Brazil and Argentina, which were sequenced after a detailed morphological analysis. Among the sequenced species, Achlya orion, Leptolegnia eccentrica, Phragmosporangium uniseriatum, and Pythiopsis irregularis are included for the first time in a phylogeny. Our results are in agreement with the recent informal proposals outlined in taxonomic overviews of the Oomycota of G.W. Beakes and collaborators, who placed the family Verrucalvaceae into the Saprolegniales and introduced the family Achlyaceae to group Achlya s.s., Brevilegnia, Dictyuchus, and Thraustotheca. These results also support the transference of Achlya androgyna to Newbya. Leptolegnia appears as paraphyletic, with the separation of L. eccentrica from the other species of this genus. In addition, Phragmosporangium, which is herein sequenced for the first time, clustered as sister to some species of Aphanomyces, including the type species, A. stellatus.