Anterior Foregut Microbiota of the Glassy-Winged Sharpshooter Explored Using Deep 16S rRNA Gene Sequencing from Individual Insects

The glassy-winged sharpshooter (GWSS) is an invasive insect species that transmits Xylella fastidiosa, the bacterium causing Pierce''s disease of grapevine and other leaf scorch diseases. X. fastidiosa has been shown to colonize the anterior foregut (cibarium and precibarium) of sharpshooters, where it may interact with other naturally-occurring bacterial species. To evaluate such interactions, a comprehensive list of bacterial species associated with the sharpshooter cibarium and precibarium is needed. Here, a survey of microbiota associated with the GWSS anterior foregut was conducted. Ninety-six individual GWSS, 24 from each of 4 locations (Bakersfield, CA; Ojai, CA; Quincy, FL; and a laboratory colony), were characterized for bacteria in dissected sharpshooter cibaria and precibaria by amplification and sequencing of a portion of the 16S rRNA gene using Illumina MiSeq technology. An average of approximately 150,000 sequence reads were obtained per insect. The most common genus detected was Wolbachia; sequencing of the Wolbachia ftsZ gene placed this strain in supergroup B, one of two Wolbachia supergroups most commonly associated with arthropods. X. fastidiosa was detected in all 96 individuals examined. By multilocus sequence typing, both X. fastidiosa subspecies fastidiosa and subspecies sandyi were present in GWSS from California and the colony; only subspecies fastidiosa was detected in GWSS from Florida. In addition to Wolbachia and X. fastidiosa, 23 other bacterial genera were detected at or above an average incidence of 0.1%; these included plant-associated microbes (Methylobacterium, Sphingomonas, Agrobacterium, and Ralstonia) and soil- or water-associated microbes (Anoxybacillus, Novosphingobium, Caulobacter, and Luteimonas). Sequences belonging to species of the family Enterobacteriaceae also were detected but it was not possible to assign these to individual genera. Many of these species likely interact with X. fastidiosa in the cibarium and precibarium.     

Analysis of the bacterial community in glassy-winged sharpshooter heads

The glassy‐winged sharpshooter (GWSS), Homalodisca vitripennis, is an important vector of various strains of Xylella fastidiosa, which cause disease in a variety of economically important plants. These diseases include citrus variegated chlorosis, oleander leaf scorch and Pierce's Disease of grapevines. Symbiotic control (SC) is a new strategy that uses symbiotic endophytes as biological control agents to antagonize or displace the pathogenic strains of X. fastidiosa. Candidate endophytes for use in SC must occupy the xylem of host plants and attach to the pre‐cibarium and cibarium of sharpshooter insects in order to have access to the pathogen. The study of the bacterial community of GWSS heads by isolation and denaturing gradient gel electrophoresis (DGGE) revealed the presence of species that may be suitable for use in SC. In addition, the results indicated that two important factors, insect age and choice of host plant, affect the composition of the bacterial community in GWSS heads. The main bacterial genera isolated as colonizers of GWSS heads were identified, using partial 16S rRNA gene sequencing, as Bacillus, Pseudomonas, Pedobacter and Methylobacterium, as well as the species Curtobacterium flaccumfaciens. DGGE patterns revealed a diversity of endophytic species able to colonize the GWSS head. The main genera isolated in culture were also identified using this technique. Principal component analysis (PCA) from polymerase chain reaction (PCR)‐DGGE patterns indicated that the bacteria inhabiting the GWSS head are similar to those found as endophytes inside the host plants, and that insect developmental stage and preferential feeding on one host plant species over another are important factors in determining the composition of the bacterial community in the GWSS head. However, a shift in host plants for a small period of time did not cause changes in the compositions of these communities.     

Retention Sites for <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> in Four Sharpshooter Vectors (Hemiptera: Cicadellidae) Analyzed by Scanning Electron Microscopy

Xylella fastidiosa is a xylem-limited bacterium that causes citrus variegated chlorosis (CVC), Pierce’s disease of grapevine, and leaf scald of coffee and plum and many other plant species. This pathogen is vectored by sharpshooter leafhoppers (Hemiptera: Cicadellidae: Cicadellinae) and resides in the insect foregut. Scanning electron microscopy was used to determine the retention sites of X. fastidiosa for the most common vector species in Brazilian citrus groves, Acrogonia citrina, Bucephalogonia xanthophis, Dilobopterus costalimai, and Oncometopia facialis. After a 48-h acquisition access period on infected citrus or plum, adult sharpshooters were kept on healthy citrus seedlings for an incubation period of 2 weeks to allow for bacterial multiplication. Then the vector heads were incubated for 24 h in a fixative and transferred into a cryoprotector liquid. Bacterial rod cells exhibiting similar X. fastidiosa morphology were found laterally attached to different regions inside the cibarial pump chamber (longitudinal groove, lateral surface, cibarial diaphragm and apodemal groove) of A. citrina, O. facialis, and D. costalimai, and polarly attached to the precibarium channel of O. facialis. Polymerase chain reactions of vector’s heads were positive for the presence of X. fastidiosa. No X. fastidiosa-like cells were detected in B. xanthophis. A different type of rod-shaped bacterium was found on B. xanthophis cibarium chamber and images suggest that the cibarium wall was degraded/digested by these bacteria. Colonization patterns of X. fastidiosa in their vectors are fundamental aspects to be explored toward understanding acquisition, adhesion, and transmission mechanisms for development of X. fastidiosa control strategies.     

Pierce's Disease of Grapevines in Taiwan: Isolation,Cultivation and Pathogenicity of Xylella fastidiosa

Characteristic symptoms of Pierce's disease (PD) in grapevines (Vitis vinifera L.) were observed in 2002 in the major grape production fields of central Taiwan. Disease severity in vineyards varied, and all investigated grape cultivars were affected. Diseased tissues were collected from fields for subsequent isolation and characterization of the causal agent of the disease (Xylella fastidiosa). Koch's postulates were fulfilled by artificially inoculating two purified PD bacteria to grape cultivars Kyoho, Honey Red and Golden Muscat. The inoculated plants developed typical leaf‐scorching symptoms, and similar disease severity developed in the three cultivars from which the bacterium was readily re‐isolated, proving that the leaf scorch of grapevines in Taiwan is caused by the fastidious X. fastidiosa. This confirmed PD of grapevines is also the first report from the Asian Continent. Phylogenetic analyses were performed by comparing the 16S rRNA gene and 16S‐23S rRNA internal transcribed spacer region (16S‐23S ITS) of 12 PD strains from Taiwan with the sequences of 13 X. fastidiosa strains from different hosts and different geographical areas. Results showed that the PD strains of Taiwan were closely related to the American X. fastidiosa grape strains but not to the pear strains of Taiwan, suggesting that the X. fastidiosa grape and pear strains of Taiwan may have evolved independently from each other.     

Shotgun metagenomics and microscopy indicate diverse cyanophytes,other bacteria,and microeukaryotes in the epimicrobiota of a northern Chilean wetland Nostoc (Cyanobacteria)

Prokaryotic Nostoc, one of the world's most conspicuous and widespread algal genera (similar to eukaryotic algae, plants, and animals) is known to support a microbiome that influences host ecological roles. Past taxonomic characterizations of surface microbiota (epimicrobiota) of free‐living Nostoc sampled from freshwater systems employed 16S rRNA genes, typically amplicons. We compared taxa identified from 16S, 18S, 23S, and 28S rRNA gene sequences filtered from shotgun metagenomic sequence and used microscopy to illuminate epimicrobiota diversity for Nostoc sampled from a wetland in the northern Chilean Altiplano. Phylogenetic analysis and rRNA gene sequence abundance estimates indicated that the host was related to Nostoc punctiforme PCC 73102. Epimicrobiota were inferred to include 18 epicyanobacterial genera or uncultured taxa, six epieukaryotic algal genera, and 66 anoxygenic bacterial genera, all having average genomic coverage ≥90X. The epicyanobacteria Geitlerinemia, Oscillatoria, Phormidium, and an uncultured taxon were detected only by 16S rRNA gene; Gloeobacter and Pseudanabaena were detected using 16S and 23S; and Phormididesmis, Neosynechococcus, Symphothece, Aphanizomenon, Nodularia, Spirulina, Nodosilinea, Synechococcus, Cyanobium, and Anabaena (the latter corroborated by microscopy), plus two uncultured cyanobacterial taxa (JSC12, O77) were detected only by 23S rRNA gene sequences. Three chlamydomonad and two heterotrophic stramenopiles genera were inferred from 18S; the streptophyte green alga Chaetosphaeridium globosum was detected by microscopy and 28S rRNA genes, but not 18S rRNA genes. Overall, >60% of epimicrobial taxa were detected by markers other than 16S rRNA genes. Some algal taxa observed microscopically were not detected from sequence data. Results indicate that multiple taxonomic markers derived from metagenomic sequence data and microscopy increase epimicrobiota detection.     

Transmission of Xylella fastidiosa by naturally infected Philaenus spumarius (Hemiptera,Aphrophoridae) to different host plants

The recent establishment of Xylella fastidiosa subspecies pauca in the southern Italian region of Apulia threatens agricultural crops and the environment. Olive is an important and widespread ancient crop in Italy and, so far, the most impacted host. The meadow spittlebug Philaenus spumarius (Hemiptera, Aphrophoridae) has been identified as a vector of X. fastidiosa in southern Italy; this species is one of the most common potential vectors in Europe. To generate disease management strategies, data on X. fastidiosa transmission by P. spumarius are necessary. Therefore, we carried out transmission experiments by using field‐collected spittlebugs in 2014 and 2015 (5 and 11 collection dates, respectively), and transferring groups of insects immediately on to recipient plants. Various host plant species were tested: olive, oleander, sweet orange, grapevine and the stone fruit rootstock GF677 (Prunus persica × Prunus amygdalus). Xylella fastidiosa was detected in all the host plants after insect plant access except for grapevine; infections to sweet orange and stone fruit were not systemic. In 2015, estimates of insect X. fastidiosa infectivity were obtained; the number of PCR‐positive P. spumarius on each plant was positively correlated with the plant infection status. The proportion of P. spumarius infected with X. fastidiosa ranged from 25% to 71% during the entire survey period. The number of X. fastidiosa cells detected in P. spumarius heads ranged from 3.5 × 10 to 4.0 × 10² (CFU equivalents), which is lower than that reported for leafhopper vectors in the Americas. These data show that field‐collected P. spumarius have high rates of X. fastidiosa infection and are competent vectors.     

Morphological and molecular characterization within 26 strains of the genus Cylindrospermum (Nostocaceae,Cyanobacteria), with descriptions of three new species

Twenty‐six strains morphologically identified as Cylindrospermum as well as the closely related taxon Cronbergia siamensis were examined microscopically as well as phylogenetically using sequence data for the 16S rRNA gene and the 16S‐23S internal transcribed spacer (ITS) region. Phylogenetic analysis of the 16S rRNA revealed three distinct clades. The clade we designate as Cylindrospermum sensu stricto contained all five of the foundational species, C. maius, C. stagnale, C. licheniforme, C. muscicola, and C. catenatum. In addition to these taxa, three species new to science in this clade were described: C. badium, C. moravicum, and C. pellucidum. Our evidence indicated that Cronbergia is a later synonym of Cylindrospermum. The phylogenetic position of Cylindrospermum within the Nostocaceae was not clearly resolved in our analyses. Cylindrospermum is unusual among cyanobacterial genera in that the morphological diversity appears to be more evident than sequence divergence. Taxa were clearly separable using morphology, but had very high percent similarity among ribosomal sequences. Given the high diversity we noted in this study, we conclude that there is likely much more diversity remaining to be described in this genus.     

Morphological and genetic diversity of Beaufort Sea diatoms with high contributions from the Chaetoceros neogracilis species complex

Seventy‐five diatom strains isolated from the Beaufort Sea (Canadian Arctic) in the summer of 2009 were characterized by light and electron microscopy (SEM and TEM), as well as 18S and 28S rRNA gene sequencing. These strains group into 20 genotypes and 17 morphotypes and are affiliated with the genera Arcocellulus, Attheya, Chaetoceros, Cylindrotheca, Eucampia, Nitzschia, Porosira, Pseudo‐nitzschia, Shionodiscus, Thalassiosira, and Synedropsis. Most of the species have a distribution confined to the northern/polar area. Chaetoceros neogracilis and Chaetoceros gelidus were the most represented taxa. Strains of C. neogracilis were morphologically similar and shared identical 18S rRNA gene sequences, but belonged to four distinct genetic clades based on 28S rRNA, ITS‐1 and ITS‐2 phylogenies. Secondary structure prediction revealed that these four clades differ in hemi‐compensatory base changes (HCBCs) in paired positions of the ITS‐2, suggesting their inability to interbreed. Reproductively isolated C. neogracilis genotypes can thus co‐occur in summer phytoplankton communities in the Beaufort Sea. C. neogracilis generally occurred as single cells but also formed short colonies. It is phylogenetically distinct from an Antarctic species, erroneously identified in some previous studies as C. neogracilis, but named here as Chaetoceros sp. This work provides taxonomically validated sequences for 20 Arctic diatom taxa, which will facilitate future metabarcoding studies on phytoplankton in this region.     

Molecular Identification and Diversity of ‘Candidatus Phytoplasma solani’ Associated with Red‐leaf Disease of Salvia miltiorrhiza in China

Reddening disease has recently been threatening Salvia miltiorrhiza in China, ranging from 30 to 50%. The main symptoms observed, such as plant stunting, inflorescence malformation, leaf reddening, fibrous roots browning, skin blackening and eventually root rot, are typically associated with phytoplasma infection. The presence of phytoplasmas was demonstrated through phytoplasma‐specific PCR, with the expected amplification (1.8 kb) from symptomatic S. miltiorrhiza plants from Shangluo, Shangzhou and Luonan fields in Shaanxi Province of China. The sequences of 16S rRNA, tuf, secY and vmp1 genes amplified from LN‐1 phytoplasma shared the closest homologies of 99%, 100%, 99% and 98% with those of the reference strain Candidatus Phytoplasma solani (subgroup 16SrXII‐A), respectively. The phylogenetic trees showed that LN‐1 phytoplasma clustered with the members of 16SrXII‐A group, including Ca. P. solani. Computer‐simulated restriction fragment length polymorphism analysis further supported this classification. Diversity analysis showed that all ‘Ca. P. solani’ strains identified from the three different regions examined shared 100% identical 16S rRNA, tuf, secY and vmp1 nucleotide sequences. To the best of our knowledge, this is the first report of phytoplasma infecting the medicinal plant of S. miltiorrhiza. The results demonstrate that ‘Ca. P. solani’ is the presumptive aetiological agent of S. miltiorrhiza reddening disease in China.     

The microbiology of Bandji,palm wine of Borassus akeassii from Burkina Faso: identification and genotypic diversity of yeasts,lactic acid and acetic acid bacteria

Aim

To investigate physicochemical characteristics and especially genotypic diversity of the main culturable micro‐organisms involved in fermentation of sap from Borassus akeassii, a newly identified palm tree from West Africa.

Methods and Results

Physicochemical characterization was performed using conventional methods. Identification of micro‐organisms included phenotyping and sequencing of: 26S rRNA gene for yeasts, 16S rRNA and gyrB genes for lactic acid bacteria (LAB) and acetic acid bacteria (AAB). Interspecies and intraspecies genotypic diversities of the micro‐organisms were screened respectively by amplification of the ITS1‐5.8S rDNA‐ITS2/16S‐23S rDNA ITS regions and repetitive sequence‐based PCR (rep‐PCR). The physicochemical characteristics of samples were: pH: 3·48–4·12, titratable acidity: 1·67–3·50 mg KOH g^?1, acetic acid: 0·16–0·37%, alcohol content: 0·30–2·73%, sugars (degrees Brix): 2·70–8·50. Yeast included mainly Saccharomyces cerevisiae and species of the genera Arthroascus, Issatchenkia, Candida, Trichosporon, Hanseniaspora, Kodamaea, Schizosaccharomyces, Trigonopsis and Galactomyces. Lactobacillus plantarum was the predominant LAB species. Three other species of Lactobacillus were also identified as well as isolates of Leuconostoc mesenteroides, Fructobacillus durionis and Streptococcus mitis. Acetic acid bacteria included nine species of the genus Acetobacter with Acetobacter indonesiensis as predominant species. In addition, isolates of Gluconobacter oxydans and Gluconacetobacter saccharivorans were also identified. Intraspecies diversity was observed for some species of micro‐organisms including four genotypes for Acet. indonesiensis, three for Candida tropicalis and Lactobacillus fermentum and two each for S. cerevisiae, Trichosporon asahii, Candida pararugosa and Acetobacter tropicalis.

Conclusion

fermentation of palm sap from B. akeassii involved multi‐yeast‐LAB‐AAB cultures at genus, species and intraspecies level.

Significance and Impact of the Study

First study describing microbiological and physicochemical characteristics of palm wine from B. akeassii. Genotypic diversity of palm wine LAB and AAB not reported before is demonstrated and this constitutes valuable information for better understanding of the fermentation which can be used to improve the product quality and develop added value by‐products.     

Spatiotemporal dynamics of the bacterial microbiota on lacustrine Cladophora glomerata (Chlorophyta)

The branched periphytic green alga Cladophora glomerata, often abundant in nearshore waters of lakes and rivers worldwide, plays important ecosystem roles, some mediated by epibiotic microbiota that benefit from host‐provided surface, organic C, and O₂. Previous microscopy and high‐throughput sequencing studies have indicated surprising epibiont taxonomic and functional diversity, but have not included adequate consideration of sample replication or the potential for spatial and temporal variation. Here, we report the results of 16S rRNA amplicon‐based phylum‐to‐genus taxonomic analysis of Cladophora‐associated bacterial epibiota sampled in replicate from three microsites and at six times during the open‐water season of 2014, from the same lake locale (Picnic Point, Lake Mendota, Dane Co., WI, USA) explored by high‐throughput sequencing studies in two previous years. Statistical methods were used to test null hypotheses that the bacterial community: (i) is homogeneous across microsites tested, and (ii) does not change over the course of a growth season or among successive years. Results indicated a dynamic microbial community that is more strongly influenced by sampling day during the growth season than by microsite variation. A surprising diversity of bacterial genera known to be associated with the key function of methane‐oxidation (methanotrophy), including relatively high‐abundance of Crenothrix, Methylomonas, Methylovulum, and Methylocaldum–showed intraseasonal and interannual variability possibly related to temperature differences, and microsite preferences possibly related to variation in methane abundance. By contrast, a core assemblage of bacterial genera seems to persist over a growth season and from year to year, possibly transmitted by a persistent attached host resting stage.     

Salivary enzymes are injected into xylem by the glassy-winged sharpshooter, a vector of Xylella fastidiosa

A few phytophagous hemipteran species such as the glassy-winged sharpshooter, Homalodisca vitripennis, (Germar), subsist entirely on xylem fluid. Although poorly understood, aspects of the insect's salivary physiology may facilitate both xylem-feeding and transmission of plant pathogens. Xylella fastidiosa is a xylem-limited bacterium that causes Pierce's disease of grape and other scorch diseases in many important crops. X. fastidiosa colonizes the anterior foregut (precibarium and cibarium) of H. vitripennis and other xylem-feeding vectors. Bacteria form a dense biofilm anchored in part by an exopolysaccharide (EPS) matrix that is reported to have a β-1,4-glucan backbone. Recently published evidence supports the following, salivation-egestion hypothesis for the inoculation of X. fastidiosa during vector feeding. The insect secretes saliva into the plant and then rapidly takes up a mixture of saliva and plant constituents. During turbulent fluid movements in the precibarium, the bacteria may become mechanically and enzymatically dislodged; the mixture is then egested back out through the stylets into plant cells, possibly including xylem vessels. The present study found that proteins extracted from dissected H. vitripennis salivary glands contain several enzyme activities capable of hydrolyzing glycosidic linkages in polysaccharides such as those found in EPS and plant cell walls, based on current information about the structures of those polysaccharides. One of these enzymes, a β-1,4-endoglucanase (EGase) was enriched in the salivary gland protein extract by subjecting the extract to a few, simple purification steps. The EGase-enriched extract was then used to generate a polyclonal antiserum that was used for immunohistochemical imaging of enzymes in sharpshooter salivary sheaths in grape. Results showed that enzyme-containing gelling saliva is injected into xylem vessels during sharpshooter feeding, in one case being carried by the transpiration stream away from the injection site. Thus, the present study provides support for the salivation-egestion hypothesis.     

Novel evidence suggests that a ‘Rickettsia felis‐like’ organism is an endosymbiont of the desert flea,Xenopsylla ramesis

Fleas are acknowledged vectors and reservoirs of various bacteria that present a wide range of pathogenicity. In this study, fleas collected from wild rodents from the Negev desert in southern Israel were tested for RickettsiaDNA by targeting the 16S rRNA (rrs) gene. Thirty‐eight Xenopsylla ramesis, 91 Synosternus cleopatrae and 15 Leptopsylla flea pools (a total of 568 fleas) were screened. RickettsiaDNA was detected in 100% of the X. ramesis and in one S. cleopatrae flea pools. None of L. algira flea pools was found positive. All positive flea pools were further characterized by sequencing of five additional genetic loci (gltA, ompB, ompA, htrA and fusA). The molecular identification of the positive samples showed all sequences to be closely related to the ‘Rickettsia felis‐like’ organisms (99–100% similarities in the six loci). To further investigate the association between ‘R. felis‐like’ and X. ramesis fleas, ten additional single X. ramesis adult fleas collected from the wild and five laboratory‐maintained X. ramesis imago, five larva pools (2–18 larvae per pool) and two egg pools (18 eggs per pool) were tested for the presence of ‘R. felis‐like’ DNA. All samples were found positive by a specific ompAPCR assay, confirming the close association of this Rickettsia species with X. ramesis in all its life stages. These results suggest a symbiotic association between ‘Rickettsia felis‐like’ and X. ramesis fleas.     

Molecular detection and characterization of a stolbur phytoplasma associated with Narcissus tazetta phyllody in Iran

During field surveys in 2015, a phytoplasma‐associated disease was identified in Narcissus tazetta plants in Behbahan, Iran. The characteristic symptoms were phyllody and virescence. The presence of phytoplasma in symptomatic plants was confirmed using PCR amplification and sequencing of 16S rRNA, tuf, secY and vmp1 genes. Based on the blastn results, the sequences of 16S rRNA, tuf, secY and vmp1 genes shared, respectively, 99%, 100%, 99% and 99% sequence identity with phytoplasma strains in 16SrXII‐A subgroup. RFLP and phylogenetic analyses using the sequences of 16S rRNA, tuf and secY genes confirmed the assortment of studied strains to 16SrXII‐A phytoplasma subgroup. Sequence comparison of these four genes revealed that all the sequences of 28 strains studied were identical. To the best of our knowledge, the association of “Candidatus Phytoplasma solani” with N. tazetta was demonstrated for the first time in the world.     

Potential vectors of Xylella fastidiosa: a study of leafhoppers and treehoppers in citrus agroecosystems affected by Citrus Variegated Chlorosis

This study investigated the predominant leafhopper and treehopper (Hemiptera, Auchenorrhyncha) species in Citrus Variegated Chlorosis (CVC)‐affected citrus agroecosystems in Argentina, their seasonal fluctuation, and their potential role as vectors of Xylella fastidiosa Wells et al., using molecular methods for detection. More than 6 000 Auchenorrhyncha were collected from three citrus agroecosystems over a period of 3 years using yellow sticky traps and entomological nets. Cicadellidae and Membracidae were the most abundant families. Of the 43 species identified, five were predominant in citrus orchards, and three were predominant in weeds surrounding citrus plants. All predominant species and another four non‐predominant species tested positive for X. fastidiosa in PCR and real‐time PCR assays. In a transmission assay, Dechacona missionum (Berg), Tapajosa rubromarginata (Signoret), and Cyphonia clavigera (Fabricius) transmitted X. fastidiosa successfully. Scaphytopius bolivianus Oman and Frequenamia spiniventris (Linnavuori) populations increased once (during the summer), possibly due to favorable weather conditions, and Bucephalogonia xanthophis (Berg), Molomea lineiceps Young, and T. rubromarginata populations increased twice a year: once in summer and once in winter, coinciding with the increase in early citrus shoots (flush). Among the X. fastidiosa‐positive species, those with the higher population densities during the sprouting period, where trees are highly susceptible to infection, must be considered as most relevant vectors of CVC in the citrus‐growing areas in Argentina.     

Transmission of Pantoea agglomerans—A paratransgenic control agent—Within a Homalodisca vitripennis population

Paratransgenic control of pests can be an alternative to chemical control, which is associated with environmental contamination, and higher input cost. We have recently developed paratransgenic control strategy to block transmission of the Pierce's disease pathogen—Xylella fastidiosa by its arthropod vector—Homalodisca vitripennis (the glassy-winged sharpshooter) using antimicrobial peptide-expressing strains of Pantoea agglomerans. In the present study, we report horizontal transmission of one of such P. agglomerans strains from H. vitripennis. This process indicates that P. agglomerans can self-sustain in a population of H. vitripennis and, thus, can provide paratransgenic control of X. fastidiosa in field. These results also provide basis for future field studies.     

Initial Genetic Analysis of Xylella fastidiosa in Texas

Xylella fastidiosa is the causative agent of Pierce’s Disease of grape. No published record of X. fastidiosa genetics in Texas exists despite growing financial risk to the U.S. grape industry, a Texas population of the glassy-winged sharpshooter insect vector (Homalodisca vitripennis) now spreading in California, and evidence that the bacterium is ubiquitous to southern states. Using sequences of conserved gyrB and mopB genes, we have established at least two strains in Texas, grape strain and ragweed strain, corresponding genetically with subsp. piercei and multiplex, respectively. The grape strain in Texas is found in Vitis vinifera varieties, hybrid vines, and wild Vitis near vineyards, whereas the ragweed strain in Texas is found in annuals, shrubs, and trees near vineyards or other areas. RFLP and QRT PCR techniques were used to differentiate grape and ragweed strains with greater efficiency than sequencing and are practical for screening numerous X. fastidiosa isolates for clade identity.     

Phylogenetic analysis of cultivation‐resistant terrestrial cyanobacteria with massive sheaths (Stigonema spp. and Petalonema alatum,Nostocales, Cyanobacteria) using single‐cell and filament sequencing of environmental samples

Molecular assessment of a large portion of traditional cyanobacterial taxa has been hindered by the failure to isolate and grow them in culture. In this study, we developed an optimized protocol for single cell/filament isolation and 16S rRNA gene sequencing of terrestrial cyanobacteria with large mucilaginous sheaths, and applied it to determine the phylogenetic position of typical members of the genera Petalonema and Stigonema. A methodology based on a glass‐capillary isolation technique and a semi‐nested PCR protocol enabled reliable sequencing of the 16S rRNA gene from all samples analyzed. Ten samples covering seven species of Stigonema from Europe, North and Central America, and Hawaii, and the type species of Petalonema from Slovakia were sequenced. Contrary to some previous studies, which proposed a relationship with heteropolar nostocalean cyanobacteria, Petalonema appeared to belong to the family Scytonemataceae. Analysis of Stigonema specimens recovered a unique coherent phylogenetic cluster, substantially broadening our knowledge of the molecular diversity within this genus. Neither the uni‐ to biseriate species nor the multiseriate species formed monophyletic subclusters within the genus. Typical multiseriate species of Stigonema clustered in a phylogenetic branch derived from uni‐ to biseriate S. ocellatum Thuret ex Bornet & Flahault in our analysis, suggesting that species with more complex thalli may have evolved from the more simple ones. We propose the technique tested in this study as a promising tool for a future revision of the molecular taxonomy in cyanobacteria.     

Onset and establishment of diazotrophs and other bacterial associates in the early life history stages of the coral Acropora millepora

Early establishment of coral–microbial symbioses is fundamental to the fitness of corals, but comparatively little is known about the onset and succession of bacterial communities in their early life history stages. In this study, bacterial associates of the coral Acropora millepora were characterized throughout the first year of life, from larvae and 1‐week‐old juveniles reared in laboratory conditions in the absence of the dinoflagellate endosymbiont Symbiodinium to field‐outplanted juveniles with established Symbiodinium symbioses, and sampled at 2 weeks and at 3, 6 and 12 months. Using an amplicon pyrosequencing approach, the diversity of both nitrogen‐fixing bacteria and of bacterial communities overall was assessed through analysis of nifH and 16S rRNA genes, respectively. The consistent presence of sequences affiliated with diazotrophs of the order Rhizobiales (23–58% of retrieved nifH sequences; 2–12% of 16S rRNA sequences), across all samples from larvae to 12‐month‐old coral juveniles, highlights the likely functional importance of this nitrogen‐fixing order to the coral holobiont. Dominance of Roseobacter‐affiliated sequences (>55% of retrieved 16S rRNA sequences) in larvae and 1‐week‐old juveniles, and the consistent presence of sequences related to Oceanospirillales and Altermonadales throughout all early life history stages, signifies their potential importance as coral associates. Increased diversity of bacterial communities once juveniles were transferred to the field, particularly of Cyanobacteria and Deltaproteobacteria, demonstrates horizontal (environmental) uptake of coral‐associated bacterial communities. Although overall bacterial communities were dynamic, bacteria with likely important functional roles remain stable throughout early life stages of Acropora millepora.     

Vessel occlusion in three cultivars of Olea europaea naturally exposed to Xylella fastidiosa in open field

Xylella fastidiosa is a Gram‐negative, xylem‐limited, bacterium which is responsible, in Italy, for the olive quick decline syndrome (OQDS). The disease is caused by the subspecies pauca and emerged a few years ago in the Apulia province of Lecce, in the Salento peninsula, on Olea europaea plants. X. fastidiosa can infect different plant species and is well known in California as the causal agent of Pierce's disease on grape. Infections of susceptible hosts with X. fastidiosa are known to result in xylem vessel occlusions, water movement impairment, and accordingly to induce the typical desiccation symptoms. In this study, we investigated xylem vessel occlusions in healthy and naturally infected O. europaea plants grown in open field by analysing three olive cultivars widespread in the region that show different degree of susceptibility to the disease: the susceptible cultivars “Ogliarola salentina” and “Cellina di Nardò,” and the tolerant cultivar “Leccino.” Our results show that occlusions were caused by tyloses and gums/pectin gels, and not by bacterial cell aggregates. Our data also indicate that occlusions are not responsible for the symptomatology of the OQDS and, as observed in Leccino plants, they are not a marker of tolerance/resistance to the disease.