Host Plant Induced Variation in Gut Bacteria of Helicoverpa armigera

Helicoverpa are important polyphagous agricultural insect pests and they have a worldwide distribution. In this study, we report the bacterial community structure in the midgut of fifth instar larvae of Helicoverpa armigera, a species prevalent in the India, China, South Asia, South East Asia, Southern & Eastern Africa and Australia. Using culturable techniques, we isolated and identified members of Bacillus firmus, Bacillus niabense, Paenibacillus jamilae, Cellulomonas variformis, Acinetobacter schindleri, Micrococcus yunnanesis, Enterobacter sp., and Enterococcus cassiliflavus in insect samples collected from host plants grown in different parts of India. Besides these the presence of Sphingomonas, Ralstonia, Delftia, Paracoccus and Bacteriodetes was determined by culture independent molecular analysis. We found that Enterobacter and Enterococcus were universally present in all our Helicoverpa samples collected from different crops and in different parts of India. The bacterial diversity varied greatly among insects that were from different host plants than those from the same host plant of different locations. This result suggested that the type of host plant greatly influences the midgut bacterial diversity of H. armigera, more than the location of the host plant. On further analyzing the leaf from which the larva was collected, it was found that the H. armigera midgut bacterial community was similar to that of the leaf phyllosphere. This finding indicates that the bacterial flora of the larval midgut is influenced by the leaf surface bacterial community of the crop on which it feeds. Additionally, we found that laboratory made media or the artificial diet is a poor bacterial source for these insects compared to a natural diet of crop plant

Xylella fastidiosa gene expression analysis by DNA microarrays

Xylella fastidiosa genome sequencing has generated valuable data by identifying genes acting either on metabolic pathways or in associated pathogenicity and virulence. Based on available information on these genes, new strategies for studying their expression patterns, such as microarray technology, were employed. A total of 2,600 primer pairs were synthesized and then used to generate fragments using the PCR technique. The arrays were hybridized against cDNAs labeled during reverse transcription reactions and which were obtained from bacteria grown under two different conditions (liquid XDM2 and liquid BCYE). All data were statistically analyzed to verify which genes were differentially expressed. In addition to exploring conditions for X. fastidiosa genome-wide transcriptome analysis, the present work observed the differential expression of several classes of genes (energy, protein, amino acid and nucleotide metabolism, transport, degradation of substances, toxins and hypothetical proteins, among others). The understanding of expressed genes in these two different media will be useful in comprehending the metabolic characteristics of X. fastidiosa, and in evaluating how important certain genes are for the functioning and survival of these bacteria in plants

Redox activities and ROS, NO and phenylpropanoids production by axenically cultured intact olive seedling roots after interaction with a mycorrhizal or a pathogenic fungus

Las raíces de las plántulas de olivo, en cultivo axénico, fueron colocadas alternativamente en contacto con Rhizophagus irregulares (micorrícicos) o con hongos Verticillim dahliae (patógenos). También se incluyeron tratamientos MeJA. Las raíces intactas (generación de anión superóxido, superóxido dismutasa y actividades de peroxidasa) se midieron en las actividades in vivo del apoplasto. Todos nuestros resultados mostraron que las actividades redox apoplásticas de raíces de las plántulas intactas en contacto con el hongo micorriza compatible fueron claramente atenuados en comparación con el hongo patógeno o tratado con MeJA, incluso en las primeras etapas usadas en el tratamiento. Los fenoles totales, flavonoides y glucósidos fenilpropanoides, también fueron cuantificados. Las raíces en contacto con el hongo micorriza no mejoraron la biosíntesis de compuestos fenólicos con respecto a los controles, mientras que los de contacto con el patógeno mejoraron de forma significativa la biosíntesis de todas las fracciones fenólicas medidas. Las especies reactivas del oxígeno y la acumulación de óxido nítrico en las raíces fueron examinadas por microscopía de fluorescencia. Todos ellas presentaron una acumulación mucho mayor en las raíces en contacto con el patógeno que con el hongo micorriza. En total, estos resultados indican que las raíces de las plántulas intactas de olivo, claramente diferenciadas entre micorrizas y hongos patógenos, atenuan las reacciones de defensa contra la primera para facilitar su creación, mientras que induce una reacción de defensa fuerte y sostenida contra el segundo. Ambas especies reactivas de oxígeno y nitrógeno parecían estar involucrados en estas respuestas desde los primeros momentos de contacto. Sin embargo, se necesitan más investigaciones para aclarar la diafonía propuesta entre ellos y sus respectivas funciones en estas respuestas ya que las imágenes de fluorescencia de las raíces revelaron que las especies reactivas del oxígeno se acumulan principalmente en el apoplasto (congruente con las actividades redox medidas en este compartimento), mientras el óxido nítrico se almacena principalmente en el citosol.Roots of intact olive seedlings, axenically cultured, were alternatively placed in contact with Rhizophagus irregularis (mycorrhizal) or Verticillim dahliae (pathogenic) fungi. MeJA treatments were also included. In vivo redox activities in the apoplast of the intact roots (anion superoxide generation, superoxide dismutase and peroxidase activities) were measured. All our results showed that apoplastic redox activities of intact seedling roots in contact with the compatible mycorrhizal fungus were clearly attenuated in comparison with the pathogenic fungus or treated with MeJA, even at the early stages of treatment used. Total phenolics, flavonoids and phenylpropanoid glycosides were also quantified. Roots in contact with the mycorrhizal fungus did not enhance the biosynthesis of phenolic compounds with respect to controls, while those in contact with the pathogenic one significantly enhanced the biosynthesis of all phenolic fractions measured. Reactive oxygen species and nitric oxid accumulation in roots were examined by fluorescence microscopy. All of them presented much higher accumulation in roots in contact with the pathogenic than with the mycorrhizal fungus. Altogether these results indicate that intact olive seedling roots clearly differentiated between mycorrhizal and pathogenic fungi, attenuating defense reactions against the first to facilitate its establishment, while inducing a strong and sustained defense reaction against the second. Both reactive oxygen and nitrogen species seemed to be involved in these responses from the first moments of contact. However, further investigations are required to clarify the proposed crosstalk between them and their respective roles in these responses since fluorescence images of roots revealed that reactive oxygen species were mainly accumulated in the apoplast (congruently with the measured redox activities in this compartment) while nitric oxid was mainly stored in the cytosol.-- Ministerio de Ciencia e Innovación. Proyecto CGL2009-12406 -- Junta de Extremadura. Proyecto PRI09A023peerReviewe

Diversity of isoprene-degrading bacteria in phyllosphere and soil communities from a high isoprene-emitting environment: a Malaysian oil palm plantation

Background: Isoprene is the most abundantly produced biogenic volatile organic compound (BVOC) on Earth, with annual global emissions almost equal to those of methane. Despite its importance in atmospheric chemistry and climate, little is known about the biological degradation of isoprene in the environment. The largest source of isoprene is terrestrial plants, and oil palms, the cultivation of which is expanding rapidly, are among the highest isoprene-producing trees. Results: DNA stable isotope probing (DNA-SIP) to study the microbial isoprene-degrading community associated with oil palm trees revealed novel genera of isoprene-utilising bacteria including Novosphingobium, Pelomonas, Rhodoblastus, Sphingomonas and Zoogloea in both oil palm soils and on leaves. Amplicon sequencing of isoA genes, which encode the α-subunit of the isoprene monooxygenase (IsoMO), a key enzyme in isoprene metabolism, confirmed that oil palm trees harbour a novel diversity of isoA sequences. In addition, metagenome assembled genomes (MAGs) were reconstructed from oil palm soil and leaf metagenomes and putative isoprene degradation genes were identified. Analysis of unenriched metagenomes showed that isoA-containing bacteria are more abundant in soils than in the oil palm phyllosphere. Conclusion: This study greatly expands the known diversity of bacteria that can metabolise isoprene and contributes to a better understanding of the biological degradation of this important but neglected climate-active gas

Gene probing reveals the widespread distribution, diversity and abundance of isoprene-degrading bacteria in the environment

Background: Approximately 500 Tg of isoprene are emitted to the atmosphere annually, an amount similar to that of methane, and despite its significant effects on the climate, very little is known about the biological degradation of isoprene in the environment. Isolation and characterisation of isoprene degraders at the molecular level has allowed the development of probes targeting isoA encoding the α-subunit of the isoprene monooxygenase. This enzyme belongs to the soluble diiron centre monooxygenase family and catalyses the first step in the isoprene degradation pathway. The use of probes targeting key metabolic genes is a successful approach in molecular ecology to study specific groups of bacteria in complex environments. Here, we developed and tested a novel isoA PCR primer set to study the distribution, abundance, and diversity of isoprene degraders in a wide range of environments. Results: The new isoA probes specifically amplified isoA genes from taxonomically diverse isoprene-degrading bacteria including members of the genera Rhodococcus, Variovorax, and Sphingopyxis. There was no cross-reactivity with genes encoding related oxygenases from non-isoprene degraders. Sequencing of isoA amplicons from DNA extracted from environmental samples enriched with isoprene revealed that most environments tested harboured a considerable variety of isoA sequences, with poplar leaf enrichments containing more phylogenetically diverse isoA genes. Quantification by qPCR using these isoA probes revealed that isoprene degraders are widespread in the phyllosphere, terrestrial, freshwater and marine environments. Specifically, soils in the vicinity of high isoprene-emitting trees contained the highest number of isoprene-degrading bacteria. Conclusion: This study provides the molecular ecology tools to broaden our knowledge of the distribution, abundance and diversity of isoprene degraders in the environment, which is a fundamental step necessary to assess the impact that microbes have in mitigating the effects of this important climate-active gas