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Identifying appropriate tissue culture and plant regeneration systems for agronomically important cultivars is often an important step in developing gene introduction techniques. Culture conditions for somatic embryogenesis and plant regeneration of 'Tifton 9' bahiagrass (Paspalum notatum Fluegge L.) from leaf-stem cross sections are described. Cultures were initiated and maintained in the dark on Schenk and Hildebrandt medium containing 3% sucrose and 6.6 mg [L.sup.-1] dicamba. Plant regeneration was initiated by transfer of callus tissue to medium lacking dicamba and incubating in continuous light. Scanning electron microscopy demonstrated somatic embryogenesis as the process leading t plantlet formation. More than 96% of 4-mm-diam. callus segments produced plants many with multiple germinating embryos. Approximately 300 plantlets were produced per gram of calli. Plants could be grown to maturity by transferring t greenhouse flats 3 wk after placing on regeneration medium. Nonspecific subculturing of primary calli resulted in a decline in regenerability and an increase in the number of albino regenerated plants after 4 to 5 mo from the initiation of callus growth on the explants. However, subculturing of specific callus cells identified as embryonic extended the efficiency of regeneration an reduced the frequency of albino regenerates. Efficiency of regenerating green plants remained essentially unchanged for 10 mo. We have therefore identified a tissue culture and plant regeneration system suitable for use in genetic engineering.

Citrus Greening or Huanglongbing (HLB) is a disease of citrus, causing high reduction in citrus production and is transmitted by the Asian citrus psyllid Diaphorina citri Kuwayama vectoring a phloem-limited bacterium Candidatus Liberibacter sp. We report research results using crowdsourcing challenge strategy identifying potential gene targets in D. citri to control the insect using RNA interference (RNAi). From 63 submitted sequences, 43 were selected and tested by feeding them to D. citri using artificial diet assays. After feeding on artificial diet, the three most effective dsRNAs causing 30% mortality above control silenced genes expressing iron-sulfur cluster subunit of the mitochondrial electron transport chain complex (Rieske), heme iron-binding terminal oxidase enzyme (Cytochrome P450) and tetrahydrobiopterin (BH4) pathway enzyme (Pterin 4α-Carbinolamine Dehydratase). These sequences were cloned into a citrus phloem-limited virus (Citrus tristeza virus , CTV T36) expressing dsRNA against these target genes in citrus. The use of a viral mediated "para-transgenic" citrus plant system caused higher mortality to adult D. citri than what was observed using artificial diet, reaching 100% when detached citrus leaves with the engineered CTV expressing dsRNA were fed to adult D. citri. Using this approach, a virus-induced gene silencing (VIGS) can be used to test future transgenic cultivars before genetically engineering citrus. RNA Seq analysis after feeding D. citri CTV-RIE on infected leaves identified transcriptionally modified genes located upstream and downstream of the targeted RIE gene. These genes were annotated showing that many are associated with the primary function of the Rieske gene that was targeted by VIGS. [ABSTRACT FROM AUTHOR]

Abstract Background Developing monocots that accumulate more vegetative tissue protein is one strategy for improving nitrogen-sequestration and nutritive value of forage and silage crops. In soybeans (a dicotyledonous legume), the vspA and B genes encode subunits of a dimeric vegetative storage protein that plays an important role in nitrogen storage in vegetative tissues. Similar genes are found in monocots; however, they do not accumulate in leaves as storage proteins, and the ability of monocot leaves to support accumulation of an ectopically expressed soybean VSP is in question. To test this, transgenic maize (Zea Mays L. Hi-II hybrid) lines were created expressing soybean vspB from a maize ubiquitin Ubi-1 promoter. Results From 81 bombardments, 101 plants were regenerated, and plants from five independent lines produced vspB transcripts and VSPβ polypeptides. In leaves from seven-week-old plants (prior to flowering), VSPβ accumulated to 0.5% of the soluble leaf protein in primary transgenic plants (R0), but to only 0.03% in R1 plants. During seed-filling (silage-stage) in R1 plants, the VSPβ protein was no longer detected in leaves and stems despite continued presence of the vspB RNA. The RNA transcripts for this peptide either became less efficiently translated, or the VSPβ protein became unstable during seed-fill. Conclusion Developmental differences in the accumulation of soybean VSPβ when transgenically expressed in maize show that despite no changes in the vspB transcript level, VSPβ protein that is readily detected in leaves of preflowering plants, becomes undetectable as seeds begin to develop.

Huanglongbing (HLB) is a deadly, incurable citrus disease putatively caused by the unculturable bacterium, 'Candidatus Liberibacter asiaticus' (CLas), and transmitted by Diaphorina citri. Prior studies suggest D. citri transmits CLas in a circulative and propagative manner; however, the precise interactions necessary for CLas transmission remain unknown, and the impact of insect sex on D. citri-CLas interactions is poorly understood despite reports of sex-dependent susceptibilities to CLas. We analyzed the transcriptome, proteome, metabolome, and microbiome of male and female adult D. citri reared on healthy or CLas-infected Citrus medica to determine shared and sex-specific responses of D. citri and its endosymbionts to CLas exposure. More sex-specific than shared D. citri responses to CLas were observed, despite there being no difference between males and females in CLas density or relative abundance. CLas exposure altered the abundance of proteins involved in immunity and cellular and oxidative stress in a sex-dependent manner. CLas exposure impacted cuticular proteins and enzymes involved in chitin degradation, as well as energy metabolism and abundance of the endosymbiont 'Candidatus Profftella armatura' in both sexes similarly. Notably, diaphorin, a toxic Profftella-derived metabolite, was more abundant in both sexes with CLas exposure. The responses reported here resulted from a combination of CLas colonization of D. citri as well as the effect of CLas infection on C. medica. Elucidating these impacts on D. citri and their endosymbionts contributes to our understanding of the HLB pathosystem and identifies the responses potentially critical to limiting or promoting CLas acquisition and propagation in both sexes. [ABSTRACT FROM AUTHOR]

A study was conducted to evaluate mosquito repellents against Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) and to expand our knowledge of the potential of essential plant oils as deterrents of ACP infestations. The psyllid vectors a bacterium, ‘ Candidatus Liberibacter asiaticus,’ presumed causal agent of a serious disease of citrus, huanglongbing (also known as citrus greening disease). Twenty-two candidate deterrents were tested as 20% solutions in methanol using a laboratory assay. Adults were free to enter and settle in one of three vials containing orange jasmine flush, one vial treated with a candidate deterrent, one vial treated with water or one treated with methanol. Seven mosquito repellents (CisPMD, DEET, DHS220, Icaridin, IR-3535, S-220, and TransPMD) and four essential oils (citronella, lavender, lemon eucalyptus, and a commercial mixture of oils from orange, clove, cinnamon, eucalyptus and rosemary) reduced ACP infestations on flush in the vials. Following a 24-h assay period, an average of 75% (range 58–88%) fewer psyllids were found in vials treated with these deterrents as compared to the combined number found in the control vials. Greenhouse evaluations were conducted on phytotoxicity of 5 or 25% solutions of eight deterrents and on ACP colonization of citrus seedlings sprayed with these deterrents. Few ACP colonized seedlings sprayed with 25% solutions, but this was attributed to phytotoxicity. Infestations were reduced on seedlings treated with 5% solutions, but these reductions also were largely attributed to phytotoxicity. Although unsprayed seedlings were in close proximity, adults found these plants and laid relatively large numbers of eggs. While some of the mosquito repellents and essential plant oils deterred infestations, none was completely effective and 5–25% solutions were too phytotoxic to be feasible as direct plant sprays. These results indicated the deterrents as tested hold little potential as a major tactic for reducing ACP infestations in citrus. Alternative application approaches to avoid phytotoxicity could be explored, and individual constituents of the more promising essential plant oils could be investigated. [ABSTRACT FROM AUTHOR]

The mitochondrial cytochrome oxidase I gene (mtCO1) and the ribosomal internal transcribed spacer 2 region (ITS2) are among the most widely used molecular markers for insect taxonomic characterization. Three economically important species of thrips, Scirtothrips dorsalis, Thrips palmi, and Frankliniella occidentalis were selected to examine the extent of intragenomic variation within these two marker regions in the family Thripidae, and determine if this variation would affect the utility of markers in thrips molecular diagnostics. For each species, intragenomic (within individual) variation and intergenomic (among individuals) variation was assessed by cloning and sequencing PCR-amplified copies. Intergenomic variation was generally higher than intragenomic variation except in cases where intergenomic variation was very low, as in mtCO1 from S. dorsalis and F. occidentalis. Intragenomic variation was detected in both markers in all three of the thrips species, however, 2-3 times more intragenomic variation was observed for ITS2 than mtCO1 in both S. dorsalis and T. palmi. Furthermore, levels of intragenomic variation were low for both of the genes in F. occidentalis. In all of the three thrips species, no sex-based clustering of haplotypes was observed in either marker. Unexpected high intragenomic variation in ITS2 for two of three thrips species did not interfere with thrips diagnostics. However, caution should be taken in applying ITS2 to certain studies of S. dorsalis and T. palmi when high levels of intragenomic variation could be problematic or confounding. In such studies, mtCO1 may be a preferable marker. Possible reasons for discrepancies in intragenomic variation among genomic regions are discussed. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) is a serious citrus disease that threatens the citrus industry worldwide. HLB is a systemic, infectious disease and the putative causal bacterium Candidatus liberibacter asiaticus (Las) resides in citrus phloem. In this study, the effects of heat treatment, chemical formulations, and application methods on bacterial titer in the citrus host, measured by quantitative PCR, were evaluated. Thermotherapy at 45 °C (Ct = 30.79 ± 6.26) was more effective against Las than thermotherapy at 40 °C (Ct = 28.72 ± 6.00) or 42 °C (Ct = 28.02 ± 6.25), and enhanced the delivery efficiency of effective chemical compounds into the citrus phloem. Ampicillin sodium (Amp) and a combination of actidione and validoxylamine A (Act + VA) were the most effective chemical formulations against Las. When Amp or Act + VA treatment was combined with thermotherapy, the area under disease progress curve standardized (AUDPCs) was significantly lower than the thermotherapy treatments alone. The Las titer of Amp and Act + VA treatment applied by bark paint were significantly reduced, compared with those of Amp or Act + VA applied by root drench. Therefore, we propose a chemo–thermotherapy strategy coupled with a bark paint application method for disease control in HLB-infected citrus plants. [ABSTRACT FROM AUTHOR]

Invasive arthropods pose unique management challenges in various environments, the first of which is correct identification. This apparently mundane task is particularly difficult if multiple species are morphologically indistinguishable but accurate identification can be determined with DNA barcoding provided an adequate reference set is available. Scirtothrips dorsalis is a highly polyphagous plant pest with a rapidly expanding global distribution and this species, as currently recognized, may be comprised of cryptic species. Here we report the development of a comprehensive DNA barcode library for S. dorsalis and seven nuclear markers via next-generation sequencing for identification use within the complex. We also report the delimitation of nine cryptic species and two morphologically distinguishable species comprising the S. dorsalis species complex using histogram analysis of DNA barcodes, Bayesian phylogenetics, and the multi-species coalescent. One member of the complex, here designated the South Asia 1 cryptic species, is highly invasive, polyphagous, and likely the species implicated in tospovirus transmission. Two other species, South Asia 2, and East Asia 1 are also highly polyphagous and appear to be at an earlier stage of global invasion. The remaining members of the complex are regionally endemic, varying in their pest status and degree of polyphagy. In addition to patterns of invasion and endemism, our results provide a framework both for identifying members of the complex based on their DNA barcode, and for future species delimiting efforts. [ABSTRACT FROM AUTHOR]

The Asian citrus psyllid, Diaphorina citri , transmits a phloem-limited bacterium, Candidatus ‘Liberibacter’ asiaticus that causes citrus greening disease. Because juvenile hormone (JH) plays an important role in adult and nymphal development, we studied the final steps in JH biosynthesis in D. citri . A putative JH acid methyltransferase ortholog gene ( jmt D) and its cognate cDNA were identified by searching D. citri genome database. Expression analysis shows expression in all life stages. In adults, it is expressed in the head-thorax, (containing the corpora allata), and the abdomen (containing ovaries and male accessory glands). A 3D protein model identified the catalytic groove with catalytically active amino acids and the S-adenosyl methionine (SAM)-binding loop. The cDNA was expressed in Escherichia coli cells and the purified enzyme showed high preference for farnesoic acid (FA) and homoFA ( k cat of 0.752 × 10 −3 and 0.217 × 10 −3 s −1 , respectively) as compared to JH acid I (JHA I) ( cis/trans/cis ; 2 Z , 6 E , 10 cis ), JHA III (2 E , 6 E , 10cis ), and JHA I ( trans / cis / cis ; 2 E , 2 Z , 10 cis ) ( k cat of 0.081 × 10 −3 , 0.013 × 10 −3 , and 0.003 × 10 −3 s −1 , respectively). This suggests that this ortholog is a Dc FA-o-methyl transferase gene ( fmt D), not a jmt D, and that JH biosynthesis in D. citri proceeds from FA to JH III through methyl farnesoate (MF). Dc FA-o-MT does not require Ca 2+ , Mg 2+ or Zn 2+ , however, Zn 2+ (1 mM) completely inhibits the enzyme probably by binding H115 at the active groove. This represents the first purified FA-o-MT from Hemiptera with preferred biological activity for FA and not JHA. [ABSTRACT FROM AUTHOR]

The bacterium Candidatus Liberibacter asiaticus (Las) has been strongly associated with huanglongbing, or citrus greening, which is one of the most devastating citrus diseases worldwide. Las is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri (Hemiptera: Psyllidae) in a persistent manner, but its interactions with the psyllid vector, particularly at the organ and cellular levels, are poorly understood. We have tested several fluorescence in situ hybridization (FISH) protocols and three nucleic acid probes for the localization of Las in haemolymph smears and dissected organs of ACP adults that fed on Las-infected plants in the field or laboratory and in sections from Las-infected citrus leaves. Las was detected by FISH and confocal laser scanning microscopy in the filter chamber, midgut, Malpighian tubules, haemolymph, salivary glands, ovaries and in muscle and fat tissues of ACP that acquired Las from infected plants, as well as in the phloem of infected citrus leaves. Las appeared as pleiomorphic bodies or short thin rods that were much more dispersed and individually distinct in citrus leaf phloem and in ACP haemolymph, but more densely aggregated in cells of the salivary glands and other ACP organs and tissues. Our results provide the first in situ demonstration of Las infection in various psyllid organs and tissues and show the near-systemic infection of ACP by Las. [ABSTRACT FROM AUTHOR]

Research progress is reported on an artificial diet for adult Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The primary objective was to develop a system for screening antimicrobial peptides and other potential toxic proteins for activity against adults. The base diet was a sterilized solution of sucrose (30%) and yellow-green food coloring (0.5%) in tap water. All of the studies presented were conducted at 25°C, 75% RH, and a photoperiod of 14:10 (L:D) h. Adult psyllids were <7 d old when they were transferred to diet. Addition of the food coloring was necessary to prompt adults to feed. Among the feeding trials discussed, a mean of 69.1 ± 3.2% adults survived for 14 d on the base sucrose diet. Survival rates of males and females were similar. Adults feeding on the sucrose diet may have ingested less food than adults feeding on citrus leaf disks based on differences in quantities of adult excrements deposited in feeding chambers. However, survival of adults feeding on leaf disks over a 2-wk period was only marginally better than survival of adults feeding on the base sucrose diet, and final rates of survival of adults fed these two food sources were hot significantly different. [ABSTRACT FROM AUTHOR]

Abstract: Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a species complex that is one of the most devastating agricultural pests worldwide and affects a broad range of food, fiber and ornamental crops. Unfortunately, using parsimony and neighbor joining methods, global phylogenetic relationships of the major races/biotypes of B. tabaci remain unresolved. Aside from the limitations of these methods, phylogenetic analyses have been limited to only small subsets of the global collection of B. tabaci, and thus limited taxon sampling has confounded the analyses. To improve our understanding of global B. tabaci phylogenetic relationships, a Bayesian phylogenetic technique was utilized to elucidate the relationships among all COI DNA sequence data available in GenBank for B. tabaci worldwide (366 specimens). As a result, the first well-resolved phylogeny for the B. tabaci species complex was produced showing 12 major well-resolved (0.70 posterior probability or above) genetic groups: B. tabaci (Mediterranean/Asia Minor/Africa), B. tabaci (Mediterranean), B. tabaci (Indian Ocean), B. tabaci (sub-Saharan Africa silverleafing), B. tabaci (Asia I), B. tabaci (Australia), B. tabaci (China), B. tabaci (Asia II), B. tabaci (Italy), B. tabaci (New World), B. tabaci (sub-Saharan Africa non-silverleafing) and B. tabaci (Uganda sweet potato). Further analysis of this phylogeny shows a close relationship of the New World B. tabaci with Asian biotypes, and characteristics of the major sub-Saharan Africa non-silverleafing clade strongly supports an African origin of B. tabaci due to its position at the base of the global phylogeny, and the diversity of well-resolved sub-clades within this group. Bayesian re-analyses of B. tabaci ITS, COI, and a combined dataset from a previous study resulted in seven major well-resolved races with high posterior probabilities, also showing the utility of the Bayesian method. Relationships of the 12 major B. tabaci genetic groups are discussed herein. [Copyright &y& Elsevier]

Presents information on a study of the `Tifton 85' bermudagrass (Cynodon dactylon (L.) Pers.) vegetative growth response to the application of ethephon (2-acid). Effects of this treatment on sebsequent vegetative; Responses of established Tifton 85 plants to ethephon treatment; Detailed information on the results.

Background: Citrus Huanglongbing (HLB) is a bacterial disease with high economic significance. The associated agent Candidatus Liberibacter asiaticus is a fastidious, phloem-limited, intracellular bacterium that is transmitted by an insect vector the Asian citrus psyllid (ACP). The genome of Ca. L. asiaticus contains protein secretion machinery that suggests host cell modulation capacity of this bacterium. Results: A total of 28 candidate effectors, an important class of secreted proteins, were predicted from the Ca. L. asiaticus genome. Sequence specific primers were designed for reverse transcription (RT) and quantitative PCR (qPCR), and expression was validated for 20 of the effector candidates in infected citrus with multiple genetic background. Using detached leaf inoculation, the mRNA of effectors was detected from 6 h to 7 days post ACP exposure. It was observed that higher bacterial titers were associated with a larger number of effectors showing amplification across all samples. The effectors' expression were compared in citrus hosts with various levels of HLB tolerance, including susceptible Duncan grapefruit and Washington navel orange, tolerant citron and Cleopatra mandarin, and resistant Pomeroy trifoliate and Carrizo citrange. Across all genotypes relatively high expression was observed for CLIBASIA_03695, CLIBASIA_00460, CLIBASIA_00420, CLIBASIA_04580, CLIBASIA_05320, CLIBASIA_04425, CLIBASIA_00525 and CLIBASIA_05315 in either a host-specific or -nonspecific manners. The two genotypes in each HLB-response group also show effector-expression profiles that seem to be different. In a companion study, the expression of effectors was compared between leaves and roots of own-rooted citrus that had been Ca. L. asiaticus-infected for more than a year. Results indicated relatively high expression of CLIBASIA_03875, CLIBASIA_04800 and CLIBASIA_05640 in all leaf and some root tissues of citron, Duncan and Cleopatra. Conclusion: This temporal and spatial expression analysis of Ca. L. asiaticus effectors identified candidates possibly critical for early bacterial colonization, host tolerance suppression and long-term survival which are all worthy of further investigation. [ABSTRACT FROM AUTHOR]

Citrus greening, also called Huanglongbing (HLB), is one of the most destructive citrus diseases worldwide. It is caused by the fastidious gram-negative α-proteobacteria bacterium Candidatus Liberibacter asiaticus (C Las) and vectored by the Asian citrus psyllid (ACP), Diaphorina citri. Currently, there is no cure for HLB, no compounds have been successful in controlling HLB, and no sustainable management practices have been established for the disease. Thus, searching for alternative citrus greening disease mitigation strategies is considered an urgent priority for a sustainable citrus industry. The aim of this study was to use compounds extracted from oak, Quercus hemisphaerica , and to assess the antibacterial effects of these against C Las-infected citrus plants. The application of aqueous oak leaf extracts showed substantial inhibitory effects against C Las in citrus plants and the activity of genes related to starch. Significant differences were also observed in plant phenotypic and physiological traits after treatments. Citrus plants treated with oak extracts displayed an increase in stomatal conductance, chlorophyll content and nutrient uptake concurrently with a reduction of C Las titer, when compared to citrus plants treated with just water. The information provided from this study suggests a new management treatment program to effectively deal with the HLB disease. • The application of aqueous oak leaf extracts showed substantial inhibitory effects against CLas. • Aqueous extract from oak improved the overall tree physiology of HLB-affected citrus. • Oak extracts represent the first potential organic cure to manage HLB. [ABSTRACT FROM AUTHOR]

Highlights: [•] JHAMT characterization. [•] Kinetic studies. [•] 3D modeling. [•] Expression in ovary and CA. [Copyright &y& Elsevier]