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)

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

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)

Salivary sheaths (unlabeled arrows) produced by psyllid adults (A–F) or nymphs (G), in the midrib or secondary veins of citrus leaves.

<p><b>A & B.</b> Salivary sheaths entering from the upper (A) or lower (B) epidermis of the leaf; note that in both cases, these sheaths entered the phloem (ph) through gaps in the fibrous ring (fr), and (in A) branching of these sheaths in the phloem and xylem tissues (arrowheads). <b>C & D</b>. Toluidine blue-stained semithin sections in the midrib showing intercellular path of salivary sheaths in the ground parenchyma (in C) and in the fibrous ring (fr) before entering the phloem (in D). <b>E</b>. Salivary sheath entering the midrib from the lower side, going through the ground parenchyma (gp), with very short branches (arrowheads) in this tissue (inset) before entering the phloem. <b>F.</b> Three salivary sheaths entering a secondary vein from the upper or lower sides, some are branched in the phloem (arrowheads) and others apparently associated with xylem vessels (arrows). <b>G</b>. Two salivary sheaths, produced by nymphs, entering from the lower side of a young citrus leaf, one (sh1) entering the vascular bundle (vb) of a secondary vein, and the other (sh2) entering the midrib from the side/corner, then going around the fibrous ring (fr) to enter the phloem (ph) through a wide gap on the side of the vascular bundle (double arrows). Leaf sections were examined via epifluorescence (A), epifluorescence with transmitted light (B, E-G), or light microscopy (C, D). Abbreviations: co, core cells; fr, fibrous ring; gp, ground parenchyma; ph, phloem; sf, salivary flange; vb, vascular bundle; xy, xylem.</p

Cross sections in the midrib or secondary veins of ‘Valencia’ orange leaves.

<p><b>A–C.</b> Sections in the midrib of mature healthy (A), mature Las-infected (B), and young Las-infected (C) leaves. <b>D & E.</b> Toluidine blue-stained semithin section (D) and confocal micrograph of a thicker (hand-cut) section (E) in the midrib of healthy leaves; single arrows indicate smaller gaps in the fibrous ring (fr); double arrows indicate wider gaps on the sides of the vascular bundle. <b>F</b>. Transmission electron micrograph showing thick-walled fibers of the fibrous ring (fr) with a small gap filled with ground parenchyma cells (gp). <b>G.</b> Section in a secondary vein of a healthy leaf. Leaf sections were examined with epifluorescence (A–C, G), light microscopy (D), confocal (E) or electron microscopy (F). Abbreviations: co, core cells; fr, fibrous ring; gp, ground parenchyma; ph, phloem; xy, xylem.</p

Percentage of <i>Diaphorina citri</i> nymphs that settled (and putatively fed) on various sites of healthy/LAS-infected young ‘Valencia’ orange leaves¹.

1<p>Results of two experiments. Means in the same row followed by the same letter are not significantly different, Ryan-Einot-Gabriel-Welsh multiple range test. Analyses conducted on arcsine-transformed percentages, raw data means presented.</p>2<p><i>F</i>_{3, 235} = 18.6, <i>P</i> = <0.0001).</p>3<p><i>F</i>_{3, 251} = 22.5, <i>P</i> = <0.0001).</p>4<p><i>F</i>_{3, 490} = 36.9, <i>P</i> = <0.0001).</p

Width of the phloem tissue and fibrous ring, and their ratio to vascular bundle diameter in the midrib and secondary veins of healthy/LAS-infected, and young or old, ‘Valencia’ orange leaves (all measurements in µm)¹.

1<p>For each comparison, means in the same column followed by the same letter are not significantly different (<i>t</i> tests, <i>P</i><0.05).</p

Differences between putative feeding sites of <i>Diaphorina citri</i> adults on healthy and LAS-infected ‘Valencia’ orange leaves¹.

1<p>For each comparison, means in the same column followed by the same letter are not significantly different, least squares means.</p>2<p><i>F</i>_{3, 12} = 77.9, <i>P</i> = <0.0001.</p>3<p><i>F</i>_{3, 12} = 48.6, <i>P</i> = <0.0001.</p>4<p><i>F</i>_{3, 12} = 92.9, <i>P</i> = <0.0001.</p>5<p><i>F</i>_{3, 12} = 165.1, <i>P</i> = <0.0001.</p

Electron micrographs of ultrathin cross sections in the stylets of a psyllid adult (A), stylets of 1^st instar nymph (B), and in the phloem of a LAS-infected citrus leaf (C).

<p>Unlabeled arrows (in C) indicate bacterial structures found in the phloem of infected leaves. Abbreviations: d, dendrites; fc, food canal; md1&2, mandibular stylets 1& 2; mx1&2, maxillary stylets 1 & 2; sc, salivary canal.</p

Mean distance to the phloem from the center (top) or sides of the midrib and in secondary veins of healthy/LAS-infected, young or old ‘Valencia’ orange leaves.

1<p>For each vein and leaf category combination, means in the same column followed by the same letter are not significantly different (<i>t</i> tests, <i>P</i><0.05).</p