Histological and cytological studies of plant infection by <Emphasis Type="Italic">Erysiphe euonymi</Emphasis>-<Emphasis Type="Italic">japonici</Emphasis>

Powdery mildew caused by Erysiphe euonymi-japonici (Eej) is an increasingly serious fungal disease on Euonymus japonicus that is an important ornamental plant. However, little is currently known about infection and pathogenesis of Eej on E. japonicus. Here, we report plant infection by Eej at the histological and cytological levels. Eej caused severe disease symptoms with white and snow-like colonies on leaf surfaces of E. japonicus. Microscopic observations were conducted continuously to define infection process of Eej on E. japonicus. Eej conidia germinated to produce appressorial germ tubes on leaf surfaces and formed irregular haustoria in plant epidermal cells at 6 h post-inoculation (hpi) and 12 hpi, respectively. After uptaking nutrients from host cells by haustoria, Eej formed numerous hyphae and extensive colonization on leaf surfaces at 96 hpi and finally produced abundant conidiophores and new conidia on leaf surfaces at 168 hpi. In addition, there was consistently a single nucleus in different Eej infection structures and haustorial development could be divided into three major stages, including formation of penetration peg, formation of haustorial neck and initial haustorium, and maturation of haustorium. These results provide useful information for further determination of Eej pathogenesis and finally controlling the disease.     

Cloning and expression analysis of the gene encoding fibrinogen-like protein A,a novel regeneration-related protein from Apostichopus japonicus

Fibrinogen-like protein A (FGLA), a member of the fibrinogen-related protein superfamily, exists in different tissues of vertebrates and invertebrates. FGLA plays crucial roles including innate immune response, blood clotting and regeneration. In this study, the fibrinogen-like protein A (fglA) was cloned from Apostichopus japonicus using rapid amplification of cDNA ends PCR techniques. The cDNA sequence of fglA is 1,524 bp with a 849 bp open reading frame encoding a polypeptide of 282 amino acids, with an N-terminal signal peptide and a conserved C-terminal domain. Bioinformatic analysis revealed that the predicted molecular weight of the whole protein is 31.9 kDa and it has an isoelectric point of 5.64. In-situ hybridization demonstrated that fglA is widely distributed in body wall, intestines, longitudinal muscles and respiratory tree. The expression levels of fglA during different regeneration stages in the body wall, intestine and respiratory trees were analyzed by real-time PCR. The expression of fglA gradually increased within 1 h in body wall, and reached a plateau before decreasing to the basal level. This indicates that fglA is associated with the regeneration of Apostichopus japonicus.     

Evidence for the Nucleo-Apical Shuttling of a Beta-Catenin Like Plasmodium falciparum Armadillo Repeat Containing Protein

Eukaryotic Armadillo (ARM) repeat proteins are multifaceted with prominent roles in cell-cell adhesion, cytoskeletal regulation and intracellular signaling among many others. One such ARM repeat containing protein, ARM Repeats Only (ARO), has recently been demonstrated in both Toxoplasma (TgARO) and Plasmodium (PfARO) parasites to be targeted to the rhoptries during the late asexual stages. TgARO has been implicated to play an important role in rhoptry positioning i.e. directing the rhoptry towards the apical end of the parasite. Here, we report for the first time that PfARO exhibits a DNA binding property and a dynamic sub-cellular localization between the nucleus (early schizont) and rhoptry (late schizont) during the different stages of the asexual blood-stage life cycle. PfARO possesses a putative nuclear export signal (NES) and the nucleo-apical shuttling was sensitive to Leptomycin B (LMB) suggesting that the nuclear export was mediated by CRM1. Importantly, PfARO specifically bound an A-T rich DNA sequence of the P. falciparum Gyrase A (PfgyrA) gene, suggesting that the DNA binding specificity of PfARO is likely due to the AT-richness of the probe. This is a novel functional characteristic that has not been reported previously for any P. falciparum ARM containing protein and suggests a putative role for PfARO in gene regulation. This study describes for the first time a conserved P. falciparum ARM repeat protein with a high degree of functional versatility.     

Molecular cloning and characterization of genistein 4′-O-glucoside specific glycosyltransferase from Bacopa monniera

Health related benefits of isoflavones such as genistein are well known. Glycosylation of genistein yields different glycosides like genistein 7-O-glycoside (genistin) and genistein 4′-O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 4′-O-glucoside from Bacopa monniera, an important Indian medicinal herb. The glycosyltransferase from B. monniera (UGT74W1) showed 49 % identity at amino acid level with the glycosyltransferases from Lycium barbarum. The UGT74W1 sequence contained all the conserved motifs present in plant glycosyltransferases. UGT74W1 was cloned in pET-30b (+) expression vector and transformed into E. coli. The molecular mass of over expressed protein was found to be around 52 kDa. Functional characterization of the enzyme was performed using different substrates. Product analysis was done using LC–MS and HPLC, which confirmed its specificity for genistein 4′-O-glucoside. Immuno-localization studies of the UGT74W1 showed its localization in the vascular bundle. Spatio-temporal expression studies under normal and stressed conditions were also performed. The control B. monniera plant showed maximum expression of UGT74W1 in leaves followed by roots and stem. Salicylic acid treatment causes almost tenfold increase in UGT74W1 expression in roots, while leaves and stem showed decrease in expression. Since salicylic acid is generated at the time of injury or wound caused by pathogens, this increase in UGT74W1 expression under salicylic acid stress might point towards its role in defense mechanism.     

Biosynthesis and hyper production of pullulan by a newly isolated strain of Aspergillus japonicus-VIT-SB1

The main focus of this study was to screen and characterize novel microbial strains isolated from culinary leaf samples, capable of producing high concentrations of pullulan. Hundred isolates were screened from the phylloplane of different plants. The results revealed that eight strains had the capability to produce exopolysaccharide (EPS) and only one potential strain (designated as VIT-SB1) could produce the significant amount of EPS (3.9 ± 0.02 %) on the 6th day of the fermentation without optimisation. The EPS synthesized by VIT-SB1 strain was confirmed to be pullulan on the basis of the results of FT-IR, HPLC and the enzymatic (Pullulanase) analysis. More than 91 % hydrolysis of pullulan by pullulanase enzyme also indicated the presence of α (1 → 6) glycosidic linkages of α (1 → 4) linked maltotriose units. This VIT-SB1 strain was identified as Aspergillus japonicus based on the nucleotide sequence of the D1/D2 domain of Large-Subunit rRNA gene. The sequence was submitted to the GenBank Nucleotide sequence database with Accession No: KC128815. This study has confirmed that pullulan production capacity of this novel strain and Aureobasidium pullulans are comparable. Hence Aspergillus japonicus-VIT-SB1 strain can be commercially exploited as a potential pullulan producing strain.     

Unraveling microbe-mediated interactions between mosquito larvae in a laboratory microcosm

Interspecies interactions have important impacts on communities and when multiple trophic levels are involved, effects can be complex and indirect. For mosquitoes, interactions experienced as larvae affect adult attributes such as survivorship, reproductive output, and longevity, factors that can affect their ability to vector disease. We examined how larvae of two ecologically distinct mosquito species, Aedes japonicus japonicus and Culex quinquefasciatus, interact at different temperatures (17 and 27 °C) and at different relative densities. We also quantified abundances of bacteria and protozoan flagellates to uncover how changes in the microbial community affect the outcome of the two mosquitoes’ interaction. At 17 °C, survival and size of both mosquito species were not affected by the other’s presence. Cx. quinquefasciatus was strongly affected by intraspecific, but not interspecific, competition at both temperatures. At 27 °C, Ae. j. japonicus larvae experienced 100 % mortality in treatments by themselves and treatments where Cx. quinquefasciatus was abundant, surviving only in the presence of low densities of Cx. quinquefasciatus. Both the total bacteria count and counts of a protozoan flagellate identified as Spumella spp. decreased with increasing numbers of Cx. quinquefasciatus. We postulate that at 27 °C, the survival of Ae. j. japonicus depends on the interaction between Cx. quinquefasciatus and the microbial community. This study demonstrates that one mosquito species may alter the microbial community in ways that indirectly influence another mosquito species’ larval survival, and by extension adult abundance and potential disease transmission.     

Identification and expression of the elongator protein 2 (Ajelp2) gene,a novel regeneration-related gene from the sea cucumber Apostichopus japonicus

Elongator proteins comprise six subunits (ELP1–ELP6) and form protein complexes. The elongator protein 2 gene (elp2) encodes a protein with a WD40 repeats domain that acts as a scaffold for complex assembly. It also plays an important role in growth and development. In this study, the full-length cDNA of elongator protein 2 (Ajelp2) was cloned from the sea cucumber Apostichopus japonicus (A. japonicus) using rapid amplification of cDNA ends PCR techniques and comprised 3,058 bp, including a 54 bp 5′ untranslated (UTR), a 526 bp 3′ UTR and a 2,478 bp open reading frame encoding a polypeptide of 825 amino acids. The Ajelp2 sequence showed high homology to 12 other species. The molecular weight and isoelectric of point the presumptive protein were 91.6 kDa and 5.84, respectively. In situ hybridization indicated that the gene is expressed in the body wall, intestine, respiratory tree and longitudinal muscle. The expression level of Ajelp2 increased in recovering of organs in sea cucumber and showed it’s the highest expression level at the 15th day in the intestine and respiratory tree. Its expression then gradually decreased to normal levels. In the body wall, the expression level of Ajelp2 was up-regulated and then down-regulated. These results indicated that Ajelp2 is involved in protein regulation during the regeneration process in the sea cucumber A. japonicus.     

Evaluating the competence of the primary vector,Culex tritaeniorhynchus,and the invasive mosquito species,Aedes japonicus japonicus,in transmitting three Japanese encephalitis virus genotypes

Japanese encephalitis virus (JEV) is maintained in an enzootic cycle between swine, water birds, and mosquitoes. JEV has circulated indigenously in Asia, with Culex tritaeniorhynchus as the primary vector. In some areas where the primary vector is scarce or absent, sporadic cases of Japanese encephalitis have been reported, with Aedes japonicus japonicus presumed to have the potential as a secondary vector. As one of the world’s most invasive culicid species, Ae. j. japonicus carries a considerable health risk for spreading diseases to wider areas, including Europe and North America. Thus, evaluation of its competency as a JEV vector, particularly in a native population, will be essential in preventing potential disease spread. In this study, the two mosquito species’ vector competence in transmitting three JEV genotypes (I, III, and V) was assessed, with Cx. tritaeniorhynchus serving as a point of reference. The mosquitoes were virus-fed and the infection rate (IR), dissemination rate (DR), and transmission rate (TR) evaluated individually by either RT-qPCR or focus forming assay. Results showed striking differences between the two species, with IR of 95% (261/274) and 9% (16/177) in Cx. tritaeniorhynchus and Ae. j. japonicus, respectively. Both mosquitoes were susceptible to all three JEV genotypes with significant differences in IR and mean viral titer. Results confirm the primary vector’s competence, but the fact that JEV was able to establish in Ae. j. japonicus is of public health significance, and with 2%–16% transmission rate it has the potential to successfully transmit JEV to the next host. This may explain the human cases and infrequent detection in primary vector-free areas. Importantly, Ae. j. japonicus could be a relevant vector spreading the disease into new areas, indicating the need for security measures in areas where the mosquito is distributed or where it may be introduced.     

Artificial reefs for sea cucumber aquaculture confirmed as settlement substrates of the moon jellyfish <Emphasis Type="Italic">Aurelia coerulea</Emphasis>

In coastal areas with a high intensity of human activities, expansion of artificial structures may enhance Aurelia spp. blooms because these constructions may provide additional substrates for the settlement and proliferation of the polyps. In the present study, the possible occurrence and distribution of Aurelia coerulea ephyrae and polyps were investigated in sea cucumber (Apostichopus japonicus) culture ponds that contain huge amounts of artificial structures. Our results showed that A. coerulea ephyrae were widely distributed in the A. japonicus culture ponds along the Bohai and Yellow Seas. Furthermore, underwater photography revealed that polyps of A. coerulea mainly occurred on the undersides of the artificial reefs made by plastic sunshade nets, tiles and substrate cages. The artificial reefs may decrease the time A. coerulea planulae spend settling, provide more hidden, calm and shady places for the settlement and proliferation of A. coerulea planulae, and thus were suitable substrates for the moon jellyfish A. coerulea. Our study suggests that the A. japonicus culture ponds may act as nursery grounds for the jellyfish A. coerulea and may potentially enhance the blooms of this species in the coastal waters along the Bohai and Yellow Seas.     

Repellence of nectar-thieving ants by a physical barrier: Adaptive role of petal hairs on Menyanthes trifoliata (Menyanthaceae)

Some plant species possess petal hairs, the defensive functions of which remain untested. I hypothesized that the petal hairs of an emergent plant species, Menyanthes trifoliata (Menyanthaceae), have a function in deterring nectar-thieving ants (Lasius japonicus). Lasius japonicus individuals were observed to stop moving forward after touching hairs and had difficulty in maintaining their balance when walking on hairs. In order to confirm the effectiveness of the hairs quantitatively, I trimmed hairs with a nose hair trimmer and then observed the behavior of flower-visiting L. japonicus. The success rate of L. japonicus in entering the floral tubes was significantly lower in the presence of intact hairs than when there were trimmed hairs. Moreover, it took L. japonicus twice as long to enter floral tubes with intact hairs, even when they succeeded. These results suggest that hairs on M. trifoliata petals are effective as a physical barrier against L. japonicus by restricting the movements of the ants.     

A saponin isolated from <Emphasis Type="Italic">Agapanthus africanus</Emphasis> differentially induces apoplastic peroxidase activity in wheat and displays fungicidal properties

A spirostane with an attached trisaccharide, (25R)-5α-spirostane-2α,3β,5α-triol 3-O-(O-α-l-rhamnopyranosyl-(1 → 2)-O-(β-d-galactopyranosyl-(1 → 3))-β-d-glucopyranoside), was isolated and identified from the aerial parts of Agapanthus africanus by activity-guided fractionation. Fungicidal properties of the crude extract, semi-purified fractions as well as the purified active saponin from A. africanus were screened in vitro against Fusarium oxysporum. At a concentration of 1 mg mL^?1, the crude extract and semi-purified ethyl acetate and dichloromethane fractions showed significant antifungal activity. The purified saponin inhibited the in vitro mycelial growth of F. oxysporum completely (100 %) at a concentration of 125 µg mL^?1. Furthermore, to verify previously observed induced resistance by crude extracts of A. africanus towards leaf rust, intercellular PR-protein activity was determined in wheat seedlings following foliar application of the purified saponin at 100 µg mL^?1. In vitro peroxidase enzyme activity increased significantly (60 %) in wheat seedlings 48 h after treatment with the purified saponin, demonstrating its role as an elicitor to activate a defence reaction in wheat.     

Attack and Success of Native and Exotic Parasitoids on Eggs of Halyomorpha halys in Three Maryland Habitats

Egg parasitoids of the exotic invasive brown marmorated stink bug, Halyomorpha halys (Stål), were investigated using lab-reared fresh (live) and frozen (killed) lab-reared sentinel egg masses deployed for 72h on foliage in three habitats—woods, orchard, and soybean field—in Maryland, USA, in summer 2014. Four native hymenopteran species, Telenomus podisi Ashmead (Scelionidae), Trissolcus euschisti (Ashmead) and Tr. brochymenae Ashmead (Scelionidae), and Anastatus reduvii (Howard) (Eupelmidae), developed and emerged from H. halys eggs. One exotic parasitoid, Trissolcus japonicus (Ashmead), emerged, providing the first known occurrence of this species in North America. Native parasitoids emerged from frozen eggs significantly more often than from fresh eggs (89.3% of egg masses and 98.1% of individual eggs), whereas the exotic Tr. japonicus did not show a similar difference, strongly suggesting adaptation to H. halys as a host by Tr. japonicus but not by the native species. Parasitoids were habitat-specific: all three Trissolcus species were significantly more likely to occur in the woods habitat, whereas Te. podisi was found exclusively in the soybean field. Further investigations are required to elucidate evolving host-parasitoid relationships, habitat specificity, and non-target effects of Tr. japonicus over the expanded range of H. halys in North America.     

3-Phosphoglycerate dehydrogenase in Mesorhizobioum loti is essential for maintaining symbiotic nitrogen fixation of Lotus japonicus root nodules

Mesorhizobium loti is a Gram negative bacterium that induces N₂-fixing root nodules on the model legume Lotus japonicus. Proteomic analysis in M. loti indicated that 3-phosphoglycerate dehydrogenase (EC. 1.1.1.95, PHGDH) protein content was 2.2 times higher in bacteroids than in cultured bacteria. A M. loti mutant (STM5) with a transposon insertion in the PHGDH gene, mll3875, showed an absolute dependence on serine or glycine in minimal medium for growth. When L. japonicus plants were infected with STM5, the roots formed nodules in numbers comparable to those formed by wild type M. loti; however, the nodules showed very low acetylene reduction activity, and significant starch granule accumulation was observed in the uninfected cells. In such nodules, vast necrosis occurred in the central tissue of the nodules, although bacteroids were detected in the infected cell of the nodules. These data indicate that serine or glycine biosynthesis by PHGDH is important for maintaining symbiosis and nitrogen fixation in L. japonicus nodules.     

JcLEA,a Novel LEA-Like Protein from Jatropha curcas,Confers a High Level of Tolerance to Dehydration and Salinity in Arabidopsis thaliana

Jatropha curcas L. is a highly drought and salt tolerant plant species that is typically used as a traditional folk medicine and biofuel crop in many countries. Understanding the molecular mechanisms that underlie the response to various abiotic environmental stimuli, especially to drought and salt stresses, in J. curcas could be important to crop improvement efforts. In this study, we cloned and characterized the gene for a late embryogenesis abundant (LEA) protein from J. curcas that we designated JcLEA. Sequence analyses showed that the JcLEA protein belongs to group 5, a subgroup of the LEA protein family. In young seedlings, expression of JcLEA is significantly induced by abscisic acid (ABA), dehydration, and salt stress. Subcellular localization analysis shows that that JcLEA protein is distributed in both the nucleus and cytoplasm. Moreover, based on growth status and physiological indices, the overexpression of JcLEA in transgenic Arabidopsis plants conferred increased resistance to both drought and salt stresses compared to the WT. Our data suggests that the group 5 JcLEA protein contributes to drought and salt stress tolerance in plants. Thus, JcLEA is a potential candidate gene for plant genetic modification.     

Mapping quantitative trait loci associated with resistance to bacterial spot (Xanthomonas arboricola pv. pruni) in peach

Bacterial spot, caused by Xanthomonas arboricola pv. pruni (Xap), is a serious disease that can affect peach fruit quality and production worldwide. This disease causes severe defoliation and blemishing of fruit, particularly in areas with high rainfall, strong winds, high humidity, and sandy soil. The molecular basis of its tolerance and susceptibility in peach is yet to be understood. An F₂ population of 63 genotypes derived from a cross between peaches “O’Henry” (susceptible) and “Clayton” (resistant) has been used for linkage map construction and quantitative trait loci (QTL) mapping. Phenotypic data for leaf and fruit response to Xap infection were collected over 2 years at two locations. A high-density genetic linkage map that covers a genetic distance of 421.4 cM with an average spacing between markers of 1.6 cM was developed using the International Peach Single Nucleotide Polymorphism Consortium (IPSC) 9K array v1. Fourteen QTLs with an additive effect on Xap resistance were detected, including four major QTLs on linkage groups (LG) 1, 4, 5, and 6. Major QTLs, Xap.Pp.OC-4.1 and Xap.Pp.OC-4.2, on LG4 were associated with Xap resistance in leaf; Xap.Pp.OC-5.1 on LG5 was associated with Xap resistance in both leaf and fruit, while Xap.Pp.OC-1.2 and Xap.Pp.OC-6.1 on LG1 and LG6, respectively, were associated with Xap resistance in fruit. This suggested separate regulation of leaf and fruit resistance for Xap in peach as well as participation of genes involved in general plant response to biotic stress. The potential for marker-assisted selection for Xap resistance in peach is discussed.     

An eceriferum locus, cer-zv, is associated with a defect in cutin responsible for water retention in barley (Hordeum vulgare) leaves

Drought limits plant growth and threatens crop productivity. A barley (Hordeum vulgare) ethylene imine-induced monogenic recessive mutant cer-zv, which is sensitive to drought, was characterized and genetically mapped in the present study. Detached leaves of cer-zv lost 34.2 % of their initial weight after 1 h of dehydration. The transpiration was much higher in cer-zv leaves than in wild-type leaves under both light and dark conditions. The stomata of cer-zv leaves functioned normally, but the cuticle of cer-zv leaves showed increased permeability to ethanol and toluidine blue dye. There was a 50–90 % reduction in four major cutin monomers, but no reduction in wax loads was found in the cer-zv mutant as compared with the wild type. Two F₂ mapping populations were established by the crosses of 23-19 × cer-zv and cer-zv × OUH602. More polymorphisms were found in EST sequences between cer-zv and OUH602 than between cer-zv and 23-19. cer-zv was located in a pericentromeric region on chromosome 4H in a 10.8 cM interval in the 23-19 × cer-zv map based on 186 gametes tested and a 1.7 cM interval in the cer-zv × OUH602 map based on 176 gametes tested. It co-segregated with EST marker AK251484 in both maps. The results indicated that the cer-zv mutant is defective in cutin, which might be responsible for the increased transpiration rate and drought sensitivity, and that the F₂ of cer-zv × OUH602 might better facilitate high resolution mapping of cer-zv.