Functional Analysis of Genes GlaDFR1 and GlaDFR2 Encoding Dihydroflavonol 4-Reductase (DFR) in Gentiana lutea L. Var. Aurantiaca (M. Laínz) M. Laínz

Anthocyanins are important pigments for flower color, determining the ornamental and economic values of horticultural plants. As a key enzyme in the biosynthesis of anthocyanidins, dihydroflavonol 4-reductase (DFR) catalyzes the reduction of dihydroflavonols to generate the precursors for anthocyanidins (i.e., leucoanthocyanidins) and anthocyanins. To investigate the functions of DFRs in plants, we cloned the GlaDFR1 and GlaDFR2 genes from the petals of Gentiana lutea var. aurantiaca and transformed both genes into Nicotiana tabacum by Agrobacterium-mediated leaf disc method. We further investigated the molecular and phenotypic characteristics of T1 generation transgenic tobacco plants selected based on the hygromycin resistance and verified by both PCR and semiquantitative real-time PCR analyses. The phenotypic segregation was observed in the flower color of the transgenic tobacco plants, showing petals darker than those in the wild-type (WT) plants. Results of high-performance liquid chromatography (HPLC) analysis showed that the contents of gentiocyanin derivatives were decreased in the petals of transgenic plants in comparison to those of WT plants. Ours results revealed the molecular functions of GlaDFR1 and GlaDFR2 in the formation of coloration, providing solid theoretical foundation and candidate genes for further genetic improvement in flower color of plants.

Expression of OsDREB2A in Transgenic Tomato Improves Drought Tolerance

Dehydration responsive element binding (DREB) are important regulatory molecules which have a crucial role in abiotic stress tolerance. The productivity of tomato, as a drought-sensitive crop, is highly restricted by drought stress. The current study aimed at introducing the OsDERB2A gene into two tomato genotypes via Agrobacterium-mediated transformation system. Cotyledonary explants were pre-cultured for two days with Agrobacterium strain LBA4404 harboring pCAMBIA1301 with OsDREB2A driven by the constitutive promoter CaMV35S for transformation. Shoots were directly regenerated on MS medium containing 1 mg l-1 zeatin and 1 mg l-1 BAP, and in presence of 30 mg l-1 hygromycin as selective agent. Only eight weeks were needed to regenerate transgenic tomato using this protocol. An OD600 of 0.4 resulted in 64.3-76.9% transformation efficiency. Stable integration and expression of the OsDREB2A gene were confirmed in transgenic tomato using PCR and RT-PCR analyses, and drought tolerance of T0 transgenic lines was confirmed by leaf disc assay in 300 mM mannitol. The superior biomass, photosynthetic pigments, free soluble sugars and proline accumulation of OsDREB2A transgenic lines over wild type in response to mannitol-stress revealed their enhanced drought tolerance and indicated that the constitutive expression of OsDREB2A might modulate the expression of other drought responsive genes.

Diversity of indigenous Bacillus thuringiensis isolates toxic to the diamondback moth, Plutella xylostella (L.) (Plutellidae: Lepidoptera)

Background Toxins from the Bacillus thuringiensis (Bt) bacterium are employed as an alternative to synthetic pesticides in pest management. The greatest threat to the long-term viability of Bt toxins is resistance evolution in the target pests. Genetic diversity and toxicity of Bt isolates were studied in this work in order to find Bt isolates with novel cry genes. Results In terms of colony morphology, among a total of 60 isolates, 51 isolates had off-white colour colonies with typical fried egg appearance, irregular shape, flat and undulate margin. Different crystal shapes, viz. spherical (88.13%), bipyramidal (49.15%), cuboidal (42.37%), rectangular, and crystals attached to spores (3.38%) were observed among Bt isolates. SDS-PAGE analysis of spore crystal mixture showed the presence of proteins with various molecular weights ranging from 124 to 26 kDa. PCR screening with cry1, cry2, cry9 and vip3A1 primers showed isolates with varied insecticidal gene combinations. Bt isolates containing cry1 genes were found to be abundant (30), followed by cry2 (9) and vip3A1 (9). Cry9 was absent in all the 60 isolates tested. Insecticidal activity of spore crystal mixtures ranged from 0 to 100% mortality. Furthermore, 12 isolates were found to be highly toxic against the larvae of diamondback moth, Plutella xylostella (L.) (Plutellidae: Lepidoptera) with 100% mortality, at 25 µg/ml in leaf disc bioassay. Conclusions The present work established the diversity of Bt isolates and confirmed the importance of continuous exploration of new Bt isolates for novel genes. Further, research needs to be carried out to unveil the hidden potential of these toxic isolates.

What Could Arrest an Eriophyoid Mite on a Plant? The Case of Aculops allotrichus from the Black Locust Tree

Aculops allotrichus is a vagrant eriophyoid that lives gregariously on the leaves of the black locust tree. This study demonstrated that conspecifics can have a significant impact on A. allotrichus females on unprofitable, old black locust leaves and can arrest them on those leaves. The effect was more pronounced in females that were exposed to artificially injured individuals than to intact ones. They not only prolonged their sojourn on leaf discs with pierced conspecifics, but also preferred the leaf disc halves with damaged individuals to clean ones. Aculops allotrichus is the first described herbivore in which artificially injured conspecifics, instead of causing alarm, keep the foraging individuals within a risky patch. Other objects, such as artificially injured or intact heterospecifics, pollen or sand, were irrelevant to the eriophyoid females on old leaf patches. In tests with old leaves of maple, magnolia and hard kiwi vine, the females postponed their movement from non-host leaf discs, which suggests that they may need more time to recognise and evaluate unfamiliar plants than familiar ones.

Rapid necrosis II: physiological and molecular analysis of 2,4-D resistance in Sumatran fleabane (Conyza sumatrensis)

In 2015, plants of Sumatran fleabane [Conyza sumatrensis (Retz.) E. Walker] were identified in a crop field with an unusual rapid necrosis herbicide symptom after application of 2,4-D. An initial study identified that the symptoms began about 2 h after herbicide application, the resistant factor is high (resistance factor = 19), and the resistance decreased at low light. The mechanism of resistance is not known yet, but the symptomatology suggests it may be related to reduced translocation, ATP-binding cassette class B (ABCB) transporters, changes on auxin perception genes or induction of genes involved in response to pathogens and abiotic stresses. The objective of this study was to investigate the mechanisms involved in the resistance to 2,4-D caused by rapid necrosis using inhibitors of enzymes involved in detoxification and carriers. Neither the inhibitors of ABCB and auxin transporters TIBA, NPA, verapamil and orthovanadate, nor the inhibitors of detoxifying enzymes, as malathion, NBD-Cl and imidazole, avoided the rapid necrosis phenotype. However, orthovanadate and sodium azide (possibly related with auxin transport) were able to partially reduce oxidative stress in leaf disc. The expression of ABCM10 (an ABCD transporter gene), TIR1_1 (an auxin receptor gene) and CAT4 (an amino acid transporter gene) was quickly reduced after 2,4-D application in the resistant accession. Contrary to our hypothesis, LESION SIMULATING DISEASE RESISTANCE 1_3 (LSD1_3) expression increased in response to 2,4-D. LSD1_3 is important for the response to pathogen and abiotic stresses. The rapid necrosis mechanism is not related to 2,4-D detoxification but might be related to changes in the TIR receptor or auxin transport. Mutations in other transporters or in proteins involved in abiotic and pathogen stresses cannot be ruled out.

Response of Different Explants for Callus Induction in Cucumber

In vitro regeneration of cucumber is relatively difficult for genetic improvement. In this regard, different concentrations of growth regulators and three types of explants (cotyledon, hypocotyl and leaf disc) were investigated for their efficiency on callus induction potential. Among different explants explored for callus induction with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), leaf disc responded earlier (4.67 days) and showed higher percentage of callus induction (91.50%) with 2 mg/l 2,4-D supplemented Murashige and Skoog (MS) media. The same concentration of 2,4-D resulted in the maximum callus fresh (0.56 g) and dry weight (0.39 g) from leaf disc explant. Then the callus was transferred to untreated, 2.0 mg/l BAP + 0.2 mg/l NAA + 1.0 mg/l Kn, 2.0 mg/l BAP + 1.0 mg/l NAA + 1.0 mg/l Kn and 2.0 mg/l BAP + 1.5 mg/l NAA + 1.0 mg/l Kn fortified MS medium. After transferring the callus of different explants to shoot regeneration media containing different concentrations of 6-benzylaminopurine (BAP), 1-naphthaleneacetic acid (NAA) and Kinetin (Kn), only cotyledon callus started to regenerate shoot. The combination of BAP (2 mg/l) + NAA (0.2 mg/l) + Kn (1 mg/l) showed highest shoot regeneration percentage (67.77%) and the maximum number of shoots (5.12) per explant were recorded in the treatment combination of 2 mg/l BAP + 0.2 mg/l NAA + 1 mg/l Kn. These results provided a basis for the optimization of the callus induction protocol of cucumber for genetic transformation.

Effects of culture medium and temperature on fungal growth of Mycena and marasmioid isolates and in vitro symbiotic culture in mycoheterotrophic orchid, Gastrodia pubilabiata

In vitro symbiotic culture of Gastrodia pubilabiata seeds were conducted with the fungal isolates from G. pubilabiata roots. We obtained five fungal isolates, which belonged to Mycena, Marasmiaceae and Omphalotaceae. Firstly, optimal temperature and culture medium for subculture of these fungal isolates were examined. All five isolates grew the fastest on malt extract agar medium. Mycelial growth rate was highest at 25 °C between 10 °C and 40 °C. Secondly, we evaluated suitable culture vessels and organic materials for symbiotic culture. Seeds germinated well in petri dishes with Quercus leaf disc on water agar medium, and the seed germination process was well observed without dense mycelium. The most developed seedlings were found in glass bottles filled with Japanese cedar leaves, but densely grew mycelium prevent accurate seedling counts. Leaves of Quercus, Japanese cedar and bamboo were used as organic materials for symbiotic culture. All three leaves induced seed germination with Mycena and Marasmiaceae fungi, but material types affected subsequent seedling growth. Our method will contribute to understanding the mycorrhizal association of Gastrodia species and also other mycoheterotrophic plants.

Critical Evaluation of Two Commercial Biocontrol Agents for Their Efficacy against B. cinerea under In Vitro and In Vivo Conditions in Relation to Different Abiotic Factors

The study evaluated the dose–response relationship of two commercial microbial biocontrol agents, Bacillus subtilis and Gliocladium catenulatum, against Botrytis cinerea both in vitro and in vivo. Inoculum doses, formulation, temperature and foliar leaf part all affected the control achieved by the two BCAs. In vitro competition assays on modified PDA plates tested a range of BCA doses (log10 3–10 CFUs or spores/droplet) at 4, 10 and 20 °C on the development of B. cinerea colonies. The dose–response relationship was influenced by both the BCA formulation and temperature. In vivo studies on lettuce plants in semi-commercial greenhouses examined the BCA dose (log10 5–9 CFUs or spores/mL) for controlling B. cinerea with a high inoculum (log10 6 spores/mL). Leaf disc assays showed that the dose–response relationship was influenced by the leaf parts sampled. These results suggest that the dose–response relationship between a BCA and specific pathogen will be significantly influenced by environmental conditions, formulation and plant phyllosplane tissue.

Gene Mining for Conserved, Non-Annotated Proteins of Podosphaera xanthii Identifies Novel Target Candidates for Controlling Powdery Mildews by Spray-Induced Gene Silencing

The powdery mildew fungus Podosphaera xanthii is one of the most important limiting factors for cucurbit production worldwide. Despite the significant efforts made by breeding and chemical companies, effective control of this pathogen remains elusive to growers. In this work, we examined the suitability of RNAi technology called spray-induced gene silencing (SIGS) for controlling cucurbit powdery mildew. Using leaf disc and cotyledon infiltration assays, we tested the efficacy of dsRNA applications to induce gene silencing in P. xanthii. Furthermore, to identify new target candidate genes, we analyzed sixty conserved and non-annotated proteins (CNAPs) deduced from the P. xanthii transcriptome in silico. Six proteins presumably involved in essential functions, specifically respiration (CNAP8878, CNAP9066, CNAP10905 and CNAP30520), glycosylation (CNAP1048) and efflux transport (CNAP948), were identified. Functional analysis of these CNAP coding genes by dsRNA-induced gene silencing resulted in strong silencing phenotypes with large reductions in fungal growth and disease symptoms. Due to their important contributions to fungal development, the CNAP1048, CNAP10905 and CNAP30520 genes were selected as targets to conduct SIGS assays under plant growth chamber conditions. The spray application of these dsRNAs induced high levels of disease control, supporting that SIGS could be a sustainable approach to combat powdery mildew diseases.