Extraction Methods Determine the Quality of Soil Microbiota Acquisition.

The soil microbiome plays a key role in plant health. Native soil microbiome inoculation, metagenomic profiling, and high-throughput cultivation require efficient microbe extraction. Sonication and oscillation are the most common methods used to extract soil microbiomes. However, the extraction efficiency of these methods has not been investigated in full. In this study, we compared the culturable microbe numbers, community structures, and alpha diversities among the different methods, including sonication, oscillation, and centrifugation, and their processing times. The study results showed that sonication significantly increases the culturable colony number compared with oscillation and centrifugation. Furthermore, the sonication strategy was found to be the main factor influencing extraction efficiency, but increased sonication time can aid in recovery from this impact. Finally, the extraction processing times were found to have a significant negative relationship with α-diversity among the extracted microbiota. In conclusion, sonication is the main factor for enriching in situ microbiota, and increased extraction time significantly decreases the α-diversity of the extracted microbiota. The results of this study provide insights into the isolation and utilization of different microorganism sources.

Quorum Sensing Is Required for the Colony Establishment of a Plant Phyllosphere Bacterium Rhodopseudomonas palustris Strain GJ-22.

The phyllosphere presents a hostile environment for many biocontrol agents; however, it is as significant as is the rhizosphere for plant health. Deploying biocontrol bacteria into the phyllosphere can efficiently suppress diseases; however, the lack of knowledge on the phyllosphere adaptive traits of biocontrol bacteria poses challenges. In this study, we demonstrated that Rhodopseudomonas palustris GJ-22 colonizes the phyllosphere by forming cell aggregates. The formation of cell aggregates required the production of exopolysaccharides (EPS), which depended on the function of the rpaI-rpaR quorum sensing (QS) mechanism, mediated by the signaling molecule p-coumaroyl-HSL (pC-HSL). The mutation of the EPS biosynthesis gene Exop1 or the signaling molecule biosynthesis gene rpaI compromised the ability of GJ-22 to tolerate reactive oxygen intermediates (ROIs), such as H2O2, in vitro and to form cell aggregates in vivo. Collectively, the results revealed that QS mediates EPS production and consequently leads to bacterial cell aggregation. IMPORTANCE Quorum sensing is used by various bacteria for coordinating the multiplication of bacterial cells in a group and for modulating the behaviors of surrounding microbial species. Host plants can benefit from this interspecies modulation, as it can disrupt the QS circuits of pathogenic bacteria. Some N-acyl homoserine lactone- (AHL-) producing bacteria that were introduced into the phyllosphere as biocontrol agents may establish AHL-based crosstalk with indigenous microbes to steer the nutritional and microecological conditions toward their own and the host plant's benefit. Here, we showed that biocontrol bacteria introduced into the phyllosphere require a functioning QS circuit to establish colonies and suppress pathogens. Furthermore, our findings provoked a broader investigation into the role of the QS circuit in beneficial microorganism-plant interactions.

Metagenomic analysis of the dynamical conversion of photosynthetic bacterial communities in different crop fields over different growth periods.

Photosynthetic bacteria are beneficial to plants, but knowledge of photosynthetic bacterial community dynamics in field crops during different growth stages is scarce. The factors controlling the changes in the photosynthetic bacterial community during plant growth require further investigation. In this study, 35 microbial community samples were collected from the seedling, flowering, and mature stages of tomato, cucumber, and soybean plants. 35 microbial community samples were assessed using Illumina sequencing of the photosynthetic reaction center subunit M (pufM) gene. The results revealed significant alpha diversity and community structure differences among the three crops at the different growth stages. Proteobacteria was the dominant bacterial phylum, and Methylobacterium, Roseateles, and Thiorhodococcus were the dominant genera at all growth stages. PCoA revealed clear differences in the structure of the microbial populations isolated from leaf samples collected from different crops at different growth stages. In addition, a dissimilarity test revealed significant differences in the photosynthetic bacterial community among crops and growth stages (P<0.05). The photosynthetic bacterial communities changed during crop growth. OTUs assigned to Methylobacterium were present in varying abundances among different sample types, which we speculated was related to the function of different Methylobacterium species in promoting plant growth development and enhancing plant photosynthetic efficiency. In conclusion, the dynamics observed in this study provide new research ideas for the detailed assessments of the relationship between photosynthetic bacteria and different growth stages of plants.

Rhodopseudomonas palustris Quorum Sensing Molecule pC-HSL Induces Systemic Resistance to TMV Infection via Upregulation of NbSIPK/NbWIPK Expressions in Nicotiana benthamiana.

G-negative bacteria produce myriad N-acyl-homoserine lactones (AHLs) that can function as quorum sensing (QS) signaling molecules. AHLs are also known to regulate various plant biological activities. p-Coumaroyl-homoserine lactone (pC-HSL) is the only QS molecule produced by a photosynthetic bacterium, Rhodopseudomonas palustris. The role of pC-HSL in the interaction between R. palustris and plant has not been investigated. In this study, we investigated the effect of pC-HSL on plant immunity and found that this QS molecule can induce a systemic resistance to Tobacco mosaic virus (TMV) infection in Nicotiana benthamiana. The results show that pC-HSL treatment can prolong the activation of two mitogen-associated protein kinase genes (i.e., NbSIPK and NbWIPK) and increase the expression of transcription factor WRKY8 as well as immune response marker genes NbPR1 and NbPR10, leading to an increased accumulation of reactive oxygen species (ROS) in the TMV-infected plants. Our results also show that pC-HSL treatment can increase activities of two ROS-scavenging enzymes, peroxidase and superoxide dismutase. Knockdown of NbSIPK or NbWIPK expression in N. benthamiana plants through virus-induced gene silencing nullified or attenuated pC-HSL-induced systemic resistance, indicating that the functioning of pC-HSL relies on the activity of those two kinases. Meanwhile, pC-HSL-pretreated plants also showed a strong induction of kinase activities of NbSIPK and NbWIPK after TMV inoculation. Taken together, our results demonstrate that pC-HSL treatment increases plant resistance to TMV infection, which is helpful to uncover the outcome of interaction between R. palustris and its host plants.

Structural characterization and functional activity of an exopolysaccharide secreted by Rhodopseudomonas palustris GJ-22.

One water exopolysaccharide, designated G-EPS, was secreted by Rhodopseudomonas palustris GJ-22 culture media. The structure of G-EPS was characterized with HPGPC, GC-MS, methylation, 1D and 2D NMR, along with UV and FT-IR spectrum. The G-EPS molecular weight was 10.026 kilodalton, and is composed of D-mannose (92.8%) and d-glucose (7.2%). The purified G-EPS promoted plant growth and induced systemic resistance against TMV in Nicotiana benthamiana. These results suggested that G-EPS is an important active component of the bio-control capacity of GJ-22.

Characterization of Rhodopseudomonas palustris population dynamics on tobacco phyllosphere and induction of plant resistance to Tobacco mosaic virus.

Although many biocontrol bacteria can be used to improve plant tolerance to stresses and to promote plant growth, the hostile environmental conditions on plant phyllosphere and the limited knowledge on bacterial colonization on plant phyllosphere minimized the beneficial effects produced by the biocontrol bacteria. Rhodopseudomonas palustris strain GJ-22 is known as a phyllosphere biocontrol agent. In this paper, we described detailed processes of strain GJ-22 colony establishment at various colonization stages. Four different types of bacterial colonies, Type 1, scattered single cells; Type 2, small cell clusters; Type 3, small cell aggregates; and Type 4, large cell aggregates, were observed in the course of bacterial colonization. We categorized bacterial colonization into four phases, which were, Phase I: bacterial colony exists as Type 1 and cell population reduced quickly; Phase II: Type 1 evolved into Type 2 and cell population remained steady; Phase III: Type 3 arose and replaced Type 2, and cell population expanded slowly; and Phase IV: Type 3 matured into Type 4 and cell population increased quickly. We have shown that the preferable location sites of bacterial aggregates on leaf phyllosphere are grooves between plant epidermal cells. Analyses of expressions of plant defence-related genes showed that, starting from Phase III, bacterial cells in the Type 3 and Type 4 colonies produced unidentified signals to induce host defence against Tobacco mosaic virus infection. In addition, we determined the crucial role of aggregates formation of GJ-22 cell on plant phyllosphere in terms of bacterial cell stress tolerance and ISR (induced systemic resistance) priming. To our knowledge, this is the first report focused on the colonization process of a phyllosphere biocontrol agent and gave a clear description on the morphological shift of bacterial colony on phyllosphere.

Silencing of Odorant-Binding Protein Gene OBP3 Using RNA Interference Reduced Virus Transmission of Tomato Chlorosis Virus.

Tomato chlorosis virus (ToCV) is widespread, seriously impacting tomato production throughout the world. ToCV is semi-persistently transmitted by Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). Currently, insect olfaction is being studied to develop novel pest control technologies to effectively control B. tabaci and whitefly-borne virus diseases. Despite current research efforts, no report has been published on the role of odorant-binding proteins (OBPs) in insect preference under the influence of plant virus. Our previous research showed that viruliferous B. tabaci preferred healthy plants at 48 h after virus acquisition. In this study, we determined the effect of OBPs on the host preference interactions of ToCV and whiteflies. Our results show that with the increase in acquisition time, the OBP gene expressions changed differently, and the OBP3 gene expression showed a trend of first rising and then falling, and reached the maximum at 48 h. These results indicate that OBP3 may participate in the host preference of viruliferous whiteflies to healthy plants. When the expression of the OBP3 gene was knocked down by an RNA interference (RNAi) technique, viruliferous Mediterranean (MED) showed no preference and the ToCV transmission rate was reduced by 83.3%. We conclude that OBP3 is involved in the detection of plant volatiles by viruliferous MED. Our results provide a theoretical basis and technical support for clarifying the transmission mechanism of ToCV by B. tabaci and could provide new avenues for controlling this plant virus and its vectors.

Nematicidal Effects of 5-Aminolevulinic Acid on Plant-Parasitic Nematodes.

Plant-parasitic nematodes are important agricultural pests and often cause serious crop losses. Novel, environmental friendly nematicides are urgently needed because of the harmful effects of some existing nematicides on human health. 5-Aminolevulinic acid (ALA) was reported as a potential biodegradable herbicide, insecticide, or plant-growth promoting agent. Lack of information on ALA against plant-parasitic nematodes prompted this investigation to determine the effects of ALA on Meloidogyne incognita, Heterodera glycines, Pratylenchus coffeae, and Bursaphelenchus xylophilus. A series of in vitro assays and one greenhouse trial were conducted to examine the nematicidal effects of ALA. The results demonstrated that ALA exhibited a strong effect of suppression against the four nematodes tested. ALA also inhibited hatching of M. incognita and H. glycines. Results from the greenhouse experiment indicated that treatment of soil with 6.0 mM ALA significantly reduced the root-gall index (RGI) and egg mass number per root system compared with the uninoculated control (P ≤ 0.05). The metabolism assays indicated that ALA treatment significantly altered the nematode metabolism including the total protein production, malondialdehyde (MDA) content, and oxidase activities. This study suggested that ALA is a promising nematicide against plant-parasitic nematodes.

Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Tylenchulus semipenetrans Using DNA Extracted from Soil.

Tylenchulus semipenetrans is an important and widespread plant-parasitic nematode of citrus worldwide and can cause citrus slow decline disease leading to significant reduction in tree growth and yield. Rapid and accurate detection of T. semipenetrans in soil is important for the disease forecasting and management. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect T. semipenetrans using DNA extracted from soil. A set of five primers was designed from the internal transcribed spacer region (ITS1) of rDNA, and was highly specific to T. semipenetrans. The LAMP reaction was performed at 63°C for 60 min. The LAMP product was visualized directly in one reaction tube by adding SYBR Green I. The detection limit of the LAMP assay was 10-2 J2/0.5 g of soil, which was 10 times more sensitive than conventional PCR (10-1 J2/0.5 g of soil). Examination of 24 field soil samples revealed that the LAMP assay was applicable to a range of soils infested naturally with T. semipenetrans, and the total assay time was less than 2.5 h. These results indicated that the developed LAMP assay is a simple, rapid, sensitive, specific and accurate technique for detection of T. semipenetrans in field soil, and contributes to the effective management of citrus slow decline disease.

Photosynthetic bacterium Rhodopseudomonas palustris GJ-22 induces systemic resistance against viruses.

Photosynthetic bacteria (PSB) have been extensively used in agriculture to promote plant growth and to improve crop quality. Their potential application in plant disease management, however, is largely overlooked. In this study, the PSB strain Rhodopseudomonas palustris GJ-22 was investigated for its ability to induce resistance against a plant virus while promoting plant growth. In the field, a foliar spray of GJ-22 suspension protected tobacco plants against tobacco mosaic virus (TMV). Under axenic conditions, GJ-22 colonized the plant phyllosphere and induced resistance against TMV. Additionally, GJ-22 produced two phytohormones, indole-3-acetic acid and 5-aminolevulinic acid, which promote growth and germination in tobacco. Furthermore, GJ-22-inoculated plants elevated their immune response under subsequent TMV infection. This research may give rise to a novel biological agent with a dual function in disease management while promoting plant growth.

Isolation of Rhp-PSP, a member of YER057c/YjgF/UK114 protein family with antiviral properties, from the photosynthetic bacterium Rhodopseudomonas palustris strain JSC-3b.

Rhodopseudomonas palustris strain JSC-3b isolated from a water canal adjacent to a vegetable field produces a protein that was purified by bioactivity-guided fractionation based on ammonium sulfate precipitation, ion-exchange absorption and size exclusion. The protein was further identified as an endoribonuclease L-PSP (Liver-Perchloric acid-soluble protein) by shotgun mass spectrometry analysis and gene identification, and it is member of YER057c/YjgF/UK114 protein family. Herein, this protein is designated Rhp-PSP. Rhp-PSP exhibited significant inhibitory activities against tobacco mosaic virus (TMV) in vivo and in vitro. To our knowledge, this represents the first report on the antiviral activity of a protein of the YER057c/YjgF/UK114 family and also the first antiviral protein isolated from R. palustris. Our research provides insight into the potential of photosynthetic bacterial resources in biological control of plant virus diseases and sustainable agriculture.

Complete genome sequence of Bacillus thuringiensis YC-10, a novel active strain against plant-parasitic nematodes.

Bacillus thuringiensis is an important microbial biopesticide for controlling agricultural pests by the production of toxic parasporal crystals proteins.Here,we report the finished annotated genome sequence of B. thuringiensis YC-10,which is highly toxic to nematodes.The complete genome sequence consists of a circular chromosome and nine circular plasmids,which the biggest plasmid harbors six parasporal crystals proteins genes consisting of cry1Aa, cry1Ac, cry1Ia, cry2Aa, cry2Ab and cryB1. The crystals proteins of Cry1Ia and Cry1Aa have high nematicidal activity against Meloidogyne incognita.

Genome Sequence of Pyrethroid-Degrading Bacterium Rhodopseudomonas palustris Strain JSC-3b.

Rhodopseudomonas palustris strain JSC-3b is a facultative, thermophilic bacterium, which was isolated from water in a canal adjacent to a vegetable field. Strain JSC-3b biodegrades several varieties of pyrethroid residues effectively through cometabolic pathways. Here, we present the genome sequence of this biodegrader.

Cometabolic biotransformation of fenpropathrin by Clostridium species strain ZP3.

A novel bacterial strain capable of degrading the pyrethroid pesticide fenpropathrin was isolated from mixed wastewater and sludge samples. Phylogenetic analysis of the 16S rDNA sequence revealed that the organism belongs to the genus Clostridium. The organism can co-metabolically transform fenpropathrin at 100 mg l(-1) at 35°C and pH 7.5 in 12 days. Metabolic products of fenpropathrin from strain ZP3 were examined by gas chromatography/mass spectrometry, and the results showed that the organism degraded fenpropathrin with an oxidization process to yield benzyl alcohol, benzenemethanol, 3,5-dimethylamphetamine. Analyses of cell-free extracts from this strain showed that the optimal degrading conditions for degrading fenpropathrin were 35°C and pH 7.5, and degradation efficiency was 20.0 mg l(-1) day(-1), and it might be potential using for rapid treating fenpropathrin, for example, on the surface of fruits and vegetables.

[Cloning and prokaryotic expression of Rhodoblastus acidophilus 5-aminolevlinate synthase gene].

5-aminolevulinic acid (ALA) is formed by the enzyme ALA synthase (ALAS). However, the fidelity of ALAS gene among species is low. The ALAS gene of photosynthetic bacteria Rhodoblastus acidophilus was cloned from its genomic DNA by conventional PCR and Veterette PCR and further sequenced. The identity of ALAS gene among photosynthetic bacteria species is from 64.0% to 95.1% according to phylogenic analysis. Furthermore, the ALAS gene was subcloned into an expression vector pQE30. For the overproduction of ALA, the recombinant ALAS was overexpressed in Escherichia coli strains JM109, M15 and BL21 (DE3), respectively. The expected 44kD protein was detected by SDS-PAGE in three E. coli strains after IPTG induction and further purified by affinity purification on Ni-NTA. The conditions including strain, medium, substrate of ALA synthesize (glycine and succinic acid), and ALA dehydratase inhibitor (levulinic acid) were optimized for attainning the maximum yield of ALA in E. coli. The ALA production was established on E. coli M15, medium 1 supplied with 100mmol/L glycine and 50mmol/L succinic acid, and 40mmol/L levulinic acid. The activity of ALAS was up to 333U/min x mg of protein. Meanwhile, the output of ALA was reached to 5.379g/L, which is the highest yield of ALA up to date by biofermentation. ALA has a variety of agricultural applications not only as an herbicide, insecticide, and growth promoting factor, but also based on its ability to confer salt and cold temperature tolerance in plants. Our recombinant bacteria are of great potential in the production of ALA. Our results offer an easy and simple ALA mass production method and may stimulate the application of ALA in agriculture.