Chia-i Hu

Bacterial volatiles promote growth in Arabidopsis

Several chemical changes in soil are associated with plant growth-promoting rhizobacteria (PGPR). Some bacterial strains directly regulate plant physiology by mimicking synthesis of plant hormones, whereas others increase mineral and nitrogen availability in the soil as a way to augment growth. Identification of bacterial chemical messengers that trigger growth promotion has been limited in part by the understanding of how plants respond to external stimuli. With an increasing appreciation of how volatile organic compounds signal plants and serve in plant defense, investigations into the role of volatile components in plant–bacterial systems now can follow. Here, we present chemical and plant-growth data showing that some PGPR release a blend of volatile components that promote growth of Arabidopsis thaliana. In particular, the volatile components 2,3-butanediol and acetoin were released exclusively from two bacterial strains that trigger the greatest level of growth promotion. Furthermore, pharmacological applications of 2,3-butanediol enhanced plant growth whereas bacterial mutants blocked in 2,3-butanediol and acetoin synthesis were devoid in this growth-promotion capacity. The demonstration that PGPR strains release different volatile blends and that plant growth is stimulated by differences in these volatile blends establishes an additional function for volatile organic compounds as signaling molecules mediating plant–microbe interactions.

Bacterial Volatiles Induce Systemic Resistance in Arabidopsis

Plant growth-promoting rhizobacteria, in association with plant roots, can trigger induced systemic resistance (ISR). Considering that low-molecular weight volatile hormone analogues such as methyl jasmonate and methyl salicylate can trigger defense responses in plants, we examined whether volatile organic compounds (VOCs) associated with rhizobacteria can initiate ISR. In Arabidopsis seedlings exposed to bacterial volatile blends from Bacillus subtilis GB03 and Bacillus amyloliquefaciens IN937a, disease severity by the bacterial pathogen Erwinia carotovora subsp. carotovora was significantly reduced compared with seedlings not exposed to bacterial volatiles before pathogen inoculation. Exposure to VOCs from rhizobacteria for as little as 4 d was sufficient to activate ISR in Arabidopsis seedlings. Chemical analysis of the bacterial volatile emissions revealed the release of a series of low-molecular weight hydrocarbons including the growth promoting VOC (2R,3R)-(-)-butanediol. Exogenous application of racemic mixture of (RR) and (SS) isomers of 2,3-butanediol was found to trigger ISR and transgenic lines of B. subtilis that emitted reduced levels of 2,3-butanediol and acetoin conferred reduced Arabidopsis protection to pathogen infection compared with seedlings exposed to VOCs from wild-type bacterial lines. Using transgenic and mutant lines of Arabidopsis, we provide evidence that the signaling pathway activated by volatiles from GB03 is dependent on ethylene, albeit independent of the salicylic acid or jasmonic acid signaling pathways. This study provides new insight into the role of bacteria VOCs as initiators of defense responses in plants.

Symptoms of Fern Distortion Syndrome Resulting from Inoculation with Opportunistic Endophytic Fluorescent Pseudomonas spp.

Background Fern Distortion Syndrome (FDS) is a serious disease of Leatherleaf fern (Rumohra adiantiformis). The main symptom of FDS is distortion of fronds, making them unmarketable. Additional symptoms include stunting, irregular sporulation, decreased rhizome diameter, and internal discoloration of rhizomes. We previously reported an association of symptoms with increased endophytic rhizome populations of fluorescent pseudomonads (FPs). The aim of the current study was to determine if FPs from ferns in Costa Rica with typical FDS symptoms would recreate symptoms of FDS. Methodology and Findings Greenhouse tests were conducted over a 29-month period. Micro-propagated ferns derived from tissue culture were first grown one year to produce rhizomes. Then, using an 8×9 randomized complete block experimental design, 8 replicate rhizomes were inoculated by dipping into 9 different treatments before planting. Treatments included water without bacteria (control), and four different groups of FPs, each at a two concentrations. The four groups of FPs included one group from healthy ferns without symptoms (another control treatment), two groups isolated from inside rhizomes of symptomatic ferns, and one group isolated from inside roots of symptomatic ferns. Symptoms were assessed 12 and 17 months later, and populations of FPs inside newly formed rhizomes were determined after 17 months. Results showed that inoculation with mixtures of FPs from ferns with FDS symptoms, but not from healthy ferns, recreated the primary symptom of frond deformities and also the secondary symptoms of irregular sporulation, decreased rhizome diameter, and internal discoloration of rhizomes. Conclusions These results suggest a model of causation of FDS in which symptoms result from latent infections by multiple species of opportunistic endophytic bacteria containing virulence genes that are expressed when populations inside the plant reach a minimum level.

Selection and Assessment of Plant Growth-Promoting Rhizobacteria for Biological Control of Multiple Plant Diseases

A study was designed to screen individual strains of plant growth-promoting rhizobacteria (PGPR) for broad-spectrum disease suppression in vitro and in planta. In a preliminary screen, 28 of 196 strains inhibited eight different tested pathogens in vitro. In a secondary screen, these 28 strains showed broad spectrum antagonistic activity to six different genera of pathogens, and 24 of the 28 strains produced five traits reported to be related to plant growth promotion, including nitrogen fixation, phosphate solubilization, indole-3-acetic acid production, siderophore production, and biofilm formation. In advanced screens, the 28 PGPR strains selected in vitro were tested in planta for biological control of multiple plant diseases including bacterial spot of tomato caused by Xanthomonas axonopodis pv. vesicatoria, bacterial speck of tomato caused by Pseudomonas syringae pv. tomato, damping-off of pepper caused by Rhizoctonia solani, and damping-off of cucumber caused by Pythium ultimum. In all, 5 of the 28 tested strains significantly reduced three of the four tested diseases, and another 19 strains showed biological control to two tested diseases. To understand the observed broad-spectrum biocontrol capacity, antiSMASH was used to predict secondary metabolite clusters of selected strains. Multiple gene clusters encoding for secondary metabolites, e.g., bacillibactin, bacilysin, and microcin, were detected in each strain. In conclusion, selected individual PGPR strains showed broad-spectrum biocontrol activity to multiple plant diseases.

Paenibacillus cucumis sp. nov. isolated from a Cucumber plant.

A Gram-positive-staining, aerobic, endospore-forming bacterial strain, isolated from the stem of a cucumber plant, was studied in detail for its taxonomic position. Based on 16S rRNA gene sequence similarity comparisons, strain AP-115T was grouped into the genus Paenibacillus, most closely related to Paenibacillus amylolyticus (98.8 %), Paenibacillus tundrae and Paenibacillus barcinonensis (both 98.4 %). The 16S rRNA gene sequence similarity to other species of the genus Paenibacillus was ≤98.4 %. Chemotaxonomic characterization supported allocation of the strain to the genus Paenibacillus. The quinone system contained exclusively menaquinone MK-7, and in the polar lipid profile diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylserine were predominating. The major component in the polyamine pattern was spermidine, and the diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. The major fatty acids were iso- and anteiso-branched fatty acids. The results of physiological and biochemical tests allowed phenotypic differentiation of strain AP-115T from closely related species. Thus, AP-115T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus cucumis sp. nov. is proposed, with AP-115T (=LMG 29222T=CCM 8653T) as the type strain.

Paenibacillus rhizoplanae sp. nov. isolated from the rhizosphere of Zea mays.

A Gram-stain-positive, aerobic, endospore-forming bacterial strain isolated from the rhizosphere of Zea mays was studied to determine its detailed taxonomic position. Based on 16S rRNA gene sequence similarity comparisons, strain JJ-64T was shown to be a member of the genus Paenibacillus, most closely related to the type strains of Paenibacillus silagei (99 %) and Paenibacillus borealis (97.5 %). 16S rRNA gene sequence similarity to all other Paenibacillus species was ≤97.5 %. DNA-DNA hybridization values to the type strains of P. silagei and P. borealis were 51 % (reciprocal 25 %) and 31 % (reciprocal 37 %), respectively. The presence of meso-diaminopimelic acid as the diagnostic diamino acid of the peptidoglycan, the major quinone MK-7 and the polyamine pattern with spermidine as the major component were well in line with the characteristics of the genus Paenibacillus. Furthermore, the polar lipid profile of strain JJ-64T with the predominant lipids diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and two unidentified aminophospholipids reflected the close phylogenetic relatedness to P. silagei. Major fatty acids were iso- and anteiso-branched components. Physiological and biochemical characteristics allowed the further phenotypic differentiation of strain JJ-64T from the most closely related species. Thus, strain JJ-64T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus rhizoplanae sp. nov. is proposed. The type strain is JJ-64T (=LMG 29875T=CCM 8725T).

Bacillus zeae sp. nov., isolated from the rhizosphere of Zea mays

A Gram-positive-staining, aerobic organism, isolated from the rhizosphere of Zea mays, was investigated in detail. Based on 16S rRNA gene sequence similarity comparisons, strain JJ-247T was grouped into the genus Bacillus, most closely related to Bacillus foraminis (98.4 %). The 16S rRNA gene sequence similarity to the sequences of the type strains of other species of the genus Bacillus was <97.4 %. The fatty acid profile with the major fatty acids, anteiso-C15 : 0, iso-C15 : 0, iso-C14 : 0 and iso-C16 : 0 supported the grouping of the strain to the genus Bacillus. The polar lipid profile contained the major components diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified aminophospholipid. The major quinone was menaquinone MK-7, and the major polyamine was spermidine. The genomic DNA G+C content of strain JJ-247T was 44.5 mol%. DNA-DNA hybridizations with the type strain B. foraminis LMG 23147T resulted in values below 70 %. In addition, physiological and biochemical test results allowed a clear phenotypic differentiation of strain JJ-247T from B. foraminis. As a consequence, JJ-247T represents a novel species of the genus Bacillus, for which we propose the name Bacillus zeae sp. nov., with JJ-247T (=CCM 8726T=LMG 29876T) as the type strain.

Mixtures of Plant-Growth-Promoting Rhizobacteria Enhance Biological Control of Multiple Plant Diseases and Plant-Growth Promotion in the Presence of Pathogens

Several studies have shown that mixtures of plant-growth-promoting rhizobacteria (PGPR) could enhance biological control activity for multiple plant diseases through the mechanisms of induced systemic resistance or antagonism. Prior experiments showed that four individual PGPR strains-AP69 (Bacillus altitudinis), AP197 (B. velezensis), AP199 (B. velezensis), and AP298 (B. velezensis)-had broad-spectrum biocontrol activity via antagonism in growth chambers against two foliar bacterial pathogens (Xanthomonas axonopodis pv. vesicatoria and Pseudomonas syringae pv. tomato) and one of two tested soilborne fungal pathogens (Rhizoctonia solani and Pythium ultimum). Based on these findings, the overall hypothesis of this study was that a mixture of two individual PGPR strains would exhibit better overall biocontrol and plant-growth promotion than the individual PGPR strains. Two separate greenhouse experiments were conducted. In each experiment, two individual PGPR strains and their mixtures were tested for biological control of three different diseases and for plant-growth promotion in the presence of the pathogens. The results demonstrated that the two individual PGPR strains and their mixtures exhibited both biological control of multiple plant diseases and plant-growth promotion. Overall, the levels of disease suppression and growth promotion were greater with mixtures than with individual PGPR strains.

Bacillus cucumis sp. nov. isolated from the rhizosphere of cucumber (Cucumis sativus).

A facultative anaerobic, Gram-positive staining, endospore-forming bacterium, isolated from the rhizosphere of cucumber (Cucumis sativus), was taxonomically investigated. Based on 16S rRNA gene sequence similarity comparisons, strain AP-6T clustered together with other species of the genus Bacillus and showed highest similarities with Bacillus drentensis LMG 21831T (99.1 %), Bacillus vireti LMG 21834T (98.7 %) and Bacillus soli LMG 21838T (98.5 %). The 16S rRNA gene sequence similarity to the sequences of the type strains of other species of the genus Bacillus was 98.5 % or less. Chemotaxonomic features supported the grouping of the strain in the genus Bacillus; for example, the major fatty acids were anteiso-C15 : 0, iso-C15 : 0 and C16 : 0, the polar lipid profile contained the major components diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified glycolipid, the major quinone was menaquinone MK-7 and the major compound in the polyamine pattern was spermidine. Additionally, DNA-DNA hybridization with B. drentensis LMG 21831T, B. vireti LMG 21834T and B. soli LMG 21838T resulted in relatedness values that were clearly below 70 %. Physiological and biochemical test results were also different from those of the most closely related species. As a consequence, AP-6T represents a novel species of the genus Bacillus, for which the name Bacillus cucumis sp. nov. is proposed, with AP-6T ( = CIP 110974T = CCM 8651T) as the type strain.

Isoptericola cucumis sp. nov., isolated from the root tissue of cucumber (Cucumis sativus).

A Gram-stain-positive, aerobic organism, showing an irregular cell morphology, was isolated from the root tissue of cucumber (Cucumis sativus) and investigated in detail for its taxonomic position. On the basis of the 16S rRNA gene sequence analysis, strain AP-38T was shown to be most closely related to Isoptericola variabilis (99.1 %) and Isoptericola nanjingensis (98.9 %). The 16S rRNA gene sequence similarity to all other species of the genus Isoptericola was ≤98.5 %. DNA-DNA relatedness to Isoptericola variablis DSM 10177T and Isoptericola nanjingensis DSM 24300T was 31(reciprocal 41 %) and 34 (reciprocal 34 %), respectively. The diagnostic diamino acid of the peptidoglycan was l-lysine. The quinone system contained predominantly menaquinones MK-9(H4) and MK-9(H2). In the polar lipid profile, major compounds were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two phosphatidylinositol mannosides. The polyamine pattern contained the major components spermidine and spermine and significant amounts of tyramine. In the fatty acid profile, anteiso-C15 : 0 and iso-C15 : 0 were present in major amounts. These data support the allocation of the strain to the genus Isoptericola. The results of physiological and biochemical characterization additionally provide phenotypic differentiation of strain AP-38T from I. variabilis and I. nanjingensis. AP-38T represents a novel species of the genus Isoptericola, for which we propose the name Isoptericola cucumis sp. nov., with AP-38T (= LMG 29223T=CCM 8653T) as the type strain.