Characterization of the Lipopolysaccharides (LPS) from Four Transposon Generated Symbiotic Mutants of Rhizobium trifolii

The LPSs from four symbiotic mutants of R. trifolii were isolated and characterized. The mutants were obtained from Dr. Barry Rolfe of The Australian National University. Three mutants have Tn5 insertions in the nod regions of the symbiotic plasmid. The fourth has a 30 kb deletion. This deletion includes the majority of the nod regions. All mutants fail to nodulate clover and fail to induce markedly curled root hairs, i.e. they are hac-nod-. The LPSs were purified as described by Carlson et al. 1978.7 The polysaccharide portion of the LPSs was purified by treating the LPSs in 1% HAc at 100° for 1 hour followed by Sephadex G-50 column chromatography. A larger and a smaller molecular weight polysaccharide (LPS1 and LPS2) are obtained. The LPS from one batch, but not from a second batch, of the insertion mutant ANU274 contains an additional polysaccharide which elutes in the void volume of the G-50 column (LPS3). Sugar compositions were determined by GC analysis of their alditol acetate derivatives. Uronic acid, 2-keto-3-deoxyoctonic acid (KDO), pyruvate and acetate were determined by colorimetric assays. Unusual sugars were identified by combined GC/MS analysis. The LPSs were also analyzed by PAGE.21 The LPS compositions from the mutants and the parent are similar, the major sugars being heptose galacturonic acid and glucuronic acid. The LPS from one batch of ANU274 was different in that it also contains rhamnose (not found in the other LPSs), increased levels of galactose and an unidentified molecule. The LPS from a second batch of ANU274 is the same as the parent and other mutant LPSs. The LPS1s from all strains, including both batches of ANU274, are very similar in composition. The LPS2s contain man, gal, glc and uronic acid, the major sugar being uronic acid. The LPS2s from all the mutants contain increased levels of glucose compared to the parent. We are uncertain if this glucose is an LPS2 component or due to contamination by small molecular weight glucans. The LPS3, present in the one batch of ANU274, contains largely rhamnose and galactose. PAGE analysis of the various LPSs show that they are very similar, except for the LPS from the one batch of ANU274. The banding patterns, except that of the one ANU274 LPS, suggest that the O-antigen may not consist of a repeating oligosaccharide but may be a single complex oligosaccharide. However, the banding pattern for the LPS from the one batch of ANU274 suggests an O-antigen which does consist of a repeating oligosaccharide. At the present time we do not know if the appearance of this different LPS in the one batch of ANU274 is due to the mutation and/or is due to slightly different growth conditions (although all batches were grown and harvested in a similar manner)

Ultrastructure of the salivary glands, alimentary canal and bacteria-like organisms in the Asian citrus psyllid, vector of citrus huanglongbing disease bacteria

AbstractThe Asian citrus psyllid (ACP, Diaphorina citri, Hemiptera: Liviidae) is the principal vector of Candidatus Liberibacter asiaticus (Las), the putative bacterial agent of citrus greening/huanglongbing (HLB); currently the most serious citrus disease worldwide. Las is transmitted in a persistent–propagative manner by ACP, and the salivary glands and midgut have been suggested as transmission barriers that can impede translocation of Las within the vector. However, no detailed ultrastructural studies have been reported on these organs in this or other psyllid species, although some bacterium-like structures have been described in them and assumed to be the causal agents of HLB. In this study, we describe the ultrastructure of the salivary glands, filter chamber, other parts of the alimentary canal, and other organs and tissues of ACP including the compound ganglionic mass (in the thorax) and the bacteriome (in the abdomen). Furthermore, in addition to two ultrastructurally apparently different symbiotic bacteria found in the bacteriome, other morphological types of bacteria were found in the gut epithelial cells and salivary glands of both Las-infected (quantitative polymerase chain reaction positive) and noninfected (quantitative polymerase chain reaction negative) ACP. These results show the importance of immunolabeling, fluorescence in situ hybridization, or other labeling techniques that must be used before identifying any bacterium-like structures in ACP or other vectors as Las or other possible agents of HLB. This ultrastructural investigation should help future work on the cellular and subcellular aspects of pathogen–psyllid relationships, including the study of receptors, binding sites, and transmission barriers of Las and other pathogens within their psyllid vectors

Unexpected High Intragenomic Variation in Two of Three Major Pest Thrips Species Does Not Affect Ribosomal Internal Transcribed Spacer 2 (ITS2) Utility for Thrips Identification

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

Use of hardwood mulch applications to improve soil characteristics of Alfisols used in Florida citrus production

IntroductionImproving soil fertility is a top priority in Florida’s citrus growing regions, especially in the age of Huanglongbing (HLB; also known as citrus greening). This disease severely reduces fine root mass, causes higher incidences of nutrient deficiencies, and eventually results in the death of affected trees. Additionally, the soils commonly found in Florida’s citrus growing regions are sandy (greater than 98%) and naturally low in fertility, making the nutrient management of HLB-affected trees even more challenging. As a result, interest in organic amendments to increase soil fertility are being tested. Although hardwood chip mulches are successfully used in other regions of the country, no studies exist observing their use on the soils in Florida’s citrus growing regions; therefore, the objectives of this study were to measure the impacts of hardwood oak mulch on (i) Florida Alfisols characteristics and (ii) HLB-affected citrus trees.MethodsA two-treatment field study using 6-year-old ‘Valencia’ sweet orange trees (Citrus × sinensis) grafted on US-812 (C. reticulata × C. trifoliata) rootstock was conducted in Florida’s Indian River District (IRD). The experimental treatment consisted of 0.08 m of hardwood chip mulch sourced from oak trees applied every September for 3 years (2020, 2021, and 2022) while the control treatment had no mulch applied. Soil chemical and physical properties, leaf nutrient concentration, and leaf Candidatus Liberibacter asiaticus (CLas) titer was collected in the fall (October), winter (January), spring (April), and summer (July).Results and discussionOverall, after 3 years, oak mulch applications increased soil available phosphorus (32%), potassium (66%), magnesium (71%), organic matter (49%), and moisture (25-88%, depending on the season); however, oak mulch inconsistently impacted leaf nutrient concentrations and was not effective at suppressing HLB. The results show that annual applications of hardwood oak mulch can improve the chemical and physical properties of sandy soils within three years, however, these improvements did not reduce the severity of HLB

Estimating Bacterial diversity in scirtothrips dorsalis (thysanoptera: thripidae) Via Next generation sequencing

The last 2 decades have produced a better understanding of insect-microbial associations and yielded some important opportunities for insect control. However, most of our knowledge comes from model systems. Thrips (Thysanoptera: Thripidae) have been understudied despite their global importance as invasive species, plant pests and disease vectors. Using a culture and primer independent next-generation sequencing and metagenomics pipeline, we surveyed the bacteria of the globally important pest, Scirtothrips dorsalis Hood. The most abundant bacterial phyla identified were Actinobacteria and Proteobacteria and the most abundant genera were Propionibacterium, Stenotrophomonas, and Pseudomonas. A total of 189 genera of bacteria were identified. The absence of any vertically transferred symbiont taxa commonly found in insects is consistent with other studies suggesting that thrips primarilly acquire resident microbes from their environment. This does not preclude a possible beneficial/intimate association between S. dorsalis and the dominant taxa identified and future work should determine the nature of these associations

Improved annotation of the insect vector of citrus greening disease: Biocuration by a diverse genomics community

The Asian citrus psyllid (Diaphorina citri Kuwayama) is the insect vector of the bacterium Candidatus Liberibacter asiaticus (CLas), the pathogen associated with citrus Huanglongbing (HLB, citrus greening). HLB threatens citrus production worldwide. Suppression or reduction of the insect vector using chemical insecticides has been the primary method to inhibit the spread of citrus greening disease. Accurate structural and functional annotation of the Asian citrus psyllid genome, as well as a clear understanding of the interactions between the insect and CLas, are required for development of new molecular-based HLB control methods. A draft assembly of the D. citri genome has been generated and annotated with automated pipelines. However, knowledge transfer from well-curated reference genomes such as that of Drosophila melanogaster to newly sequenced ones is challenging due to the complexity and diversity of insect genomes. To identify and improve gene models as potential targets for pest control, we manually curated several gene families with a focus on genes that have key functional roles in D. citri biology and CLas interactions. This community effort produced 530 manually curated gene models across developmental, physiological, RNAi regulatory and immunity-related pathways. As previously shown in the pea aphid, RNAi machinery genes putatively involved in the microRNA pathway have been specifically duplicated. A comprehensive transcriptome enabled us to identify a number of gene families that are either missing or misassembled in the draft genome. In order to develop biocuration as a training experience, we included undergraduate and graduate students from multiple institutions, as well as experienced annotators from the insect genomics research community. The resulting gene set (OGS v1.0) combines both automatically predicted and manually curated gene models.Peer reviewedBiochemistry and Molecular BiologyEntomology and Plant Patholog

Characterization of the Lipopolysaccharides (LPS) from Four Transposon Generated Symbiotic Mutants of Rhizobium trifolii

The LPSs from four symbiotic mutants of R. trifolii were isolated and characterized. The mutants were obtained from Dr. Barry Rolfe of The Australian National University. Three mutants have Tn5 insertions in the nod regions of the symbiotic plasmid. The fourth has a 30 kb deletion. This deletion includes the majority of the nod regions. All mutants fail to nodulate clover and fail to induce markedly curled root hairs, i.e. they are hac-nod-. The LPSs were purified as described by Carlson et al. 1978.7 The polysaccharide portion of the LPSs was purified by treating the LPSs in 1% HAc at 100° for 1 hour followed by Sephadex G-50 column chromatography. A larger and a smaller molecular weight polysaccharide (LPS1 and LPS2) are obtained. The LPS from one batch, but not from a second batch, of the insertion mutant ANU274 contains an additional polysaccharide which elutes in the void volume of the G-50 column (LPS3). Sugar compositions were determined by GC analysis of their alditol acetate derivatives. Uronic acid, 2-keto-3-deoxyoctonic acid (KDO), pyruvate and acetate were determined by colorimetric assays. Unusual sugars were identified by combined GC/MS analysis. The LPSs were also analyzed by PAGE.21 The LPS compositions from the mutants and the parent are similar, the major sugars being heptose galacturonic acid and glucuronic acid. The LPS from one batch of ANU274 was different in that it also contains rhamnose (not found in the other LPSs), increased levels of galactose and an unidentified molecule. The LPS from a second batch of ANU274 is the same as the parent and other mutant LPSs. The LPS1s from all strains, including both batches of ANU274, are very similar in composition. The LPS2s contain man, gal, glc and uronic acid, the major sugar being uronic acid. The LPS2s from all the mutants contain increased levels of glucose compared to the parent. We are uncertain if this glucose is an LPS2 component or due to contamination by small molecular weight glucans. The LPS3, present in the one batch of ANU274, contains largely rhamnose and galactose. PAGE analysis of the various LPSs show that they are very similar, except for the LPS from the one batch of ANU274. The banding patterns, except that of the one ANU274 LPS, suggest that the O-antigen may not consist of a repeating oligosaccharide but may be a single complex oligosaccharide. However, the banding pattern for the LPS from the one batch of ANU274 suggests an O-antigen which does consist of a repeating oligosaccharide. At the present time we do not know if the appearance of this different LPS in the one batch of ANU274 is due to the mutation and/or is due to slightly different growth conditions (although all batches were grown and harvested in a similar manner)

Bactericidal Properties of Proline-Rich <i>Aedes aegypti</i> Trypsin Modulating Oostatic Factor (<i>Aea</i>TMOF)

The antimicrobial properties of proline-rich Aedes aegypti decapeptide TMOF (AeaTMOF) and oncocin112 (1–13) were compared. Incubations with multidrug-resistant Escherichia coli cells showed that AeaTMOF (5 mM) was able to completely inhibit bacterial cell growth, whereas oncocin112 (1–13) (20 mM) partially inhibited bacterial growth as compared with bacterial cells that were not multidrug-resistant cells. AeaTMOF (5 mM) was very effective against Acinetobacter baumannii and Pseudomonas aeruginosa, completely inhibiting cell growth during 15 h incubations. AeaTMOF (5 mM) completely inhibited the Gram-positive bacteria Staphylococcus aureus and Bacillus thurengiensis sups. Israelensis cell growth, whereas oncocin112 (1–13) (10 and 20 mM) failed to affect bacterial cell growth. E. coli cells that lack the SbmA transporter were inhibited by AeaTMOF (5 mM) and not by oncocin112 (1–13) (10 to 20 mM), indicating that AeaTMOF can use other bacterial transporters than SbmA that is mainly used by proline-rich antimicrobial peptides. Incubation of E. coli cells with NaAzide showed that AeaTMOF does not use ABC-like transporters that use ATP hydrolysis to import molecules into bacterial cells. Three-dimensional modeling and docking of AeaTMOF to SbmA and MdtM transporters showed that AeaTMOF can bind these proteins, and the binding location of AeaTMOF inside these protein transporters allows AeaTMOF to be transported into the bacterial cytosol. These results show that AeaTMOF can be used as a future antibacterial agent against both multidrug-resistant Gram-positive and -negative bacteria

Stylet morphometrics and citrus leaf vein structure in relation to feeding behavior of the Asian citrus psyllid Diaphorina citri, vector of citrus huanglongbing bacterium.

The Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Psyllidae), is the primary vector of the phloem-limited bacterium Candidatus Liberibacter asiaticus (LAS) associated with huanglongbing (HLB, citrus greening), considered the world's most serious disease of citrus. Stylet morphometrics of ACP nymphs and adults were studied in relation to citrus vein structure and to their putative (histologically verified) feeding sites on Valencia orange leaves. ACP nymphs preferred to settle and feed on the lower (abaxial) side of young leaves either on secondary veins or on the sides of the midrib, whereas adults preferred to settle and feed on the upper (adaxial) or lower secondary veins of young or old leaves. Early instar nymphs can reach and probe the phloem probably because the distance to the phloem is considerably shorter in younger than in mature leaves, and is shorter from the sides of the midrib compared to that from the center. Additionally, the thick-walled 'fibrous ring' (sclerenchyma) around the phloem, which may act as a barrier to ACP stylet penetration into the phloem, is more prominent in older than in younger leaves and in the center than on the sides of the midrib. The majority (80-90%) of the salivary sheath termini produced by ACP nymphs and adults that reached a vascular bundle were associated with the phloem, whereas only 10-20% were associated with xylem vessels. Ultrastructural studies on ACP stylets and LAS-infected leaves suggested that the width of the maxillary food canal in first instar nymphs is wide enough for LAS bacteria to traverse during food ingestion (and LAS acquisition). However, the width of the maxillary salivary canal in these nymphs may not be wide enough to accommodate LAS bacteria during salivation (and LAS inoculation) into host plants. This may explain the inability of early instar nymphs to transmit LAS/HLB in earlier reports