Development of label-free electrochemical OMP-DNA probe biosensor as a highly sensitive system to detect of citrus huanglongbing.

The fabrication of the first label-free electrochemical DNA probe biosensor for highly sensitive detection of Candidatus Liberibacter asiaticus (CLas), as the causal agent of citrus huanglongbing disease, is conducted here. An OMP probe was designed based on the hybridization with its target-specific sequence in the outer membrane protein (OMP) gene of CLas. The characterization of the steps of biosensor fabrication and hybridization process between the immobilized OMP-DNA probe and the target ssDNA oligonucleotides (OMP-complementary and three mismatches OMP or OMP-mutation) was monitored using cyclic voltammetry and electrochemical impedance spectroscopy based on increasing or decreasing in the electron transfer in [Fe (CN)6]3-/4- on the modified gold electrode surface. The biosensor sensitivity indicated that the peak currents were linear over ranges from 20 to 100 nM for OMP-complementary with the detection limit of 0.026 nM (S/N = 3). The absence of any cross-interference with other biological DNA sequences confirmed a high selectivity of fabricated biosensor. Likewise, it showed good specificity in discriminating the mutation oligonucleotides from complementary target DNAs. The functional performance of optimized biosensor was achieved via the hybridization of OMP-DNA probe with extracted DNA from citrus plant infected with CLas. Therefore, fabricated biosensor indicates promise for sensitivity and early detection of citrus huanglongbing disease.

Natural Infection of Murraya paniculata and Murraya sumatrana with 'Candidatus Liberibacter asiaticus' in Java.

The phloem-limited bacterium 'Candidatus Liberibacter asiaticus' (CLas) is the putative causal pathogen of the severe Asiatic form of huanglongbing (citrus greening) and is most commonly transmitted by the Asiatic citrus psyllid Diaphorina citri. CLas severely affects many Citrus species and hybrids and has been recorded in the Citrus relative, orange jasmine, Murraya paniculata (L.) Jack (syn. M. exotica L.). In this study, 13 accessions of three Murraya species (M. paniculata, M. sumatrana Roxb., and M. lucida [G.Forst.] Mabb.) and the Papuan form of a putative hybrid (M. omphalocarpa Hayata) were identified morphologically and molecularly based on sequence identity of the matK-5'trnK region of the chloroplast genome, and infection on these plants under field conditions was determined by PCR and quantitative real-time PCR (qPCR) on two to four occasions over 14 months. CLas was repeatedly detected in leaflet midribs by PCR and qPCR on four and three accessions of M. paniculata and M. sumatrana, respectively. It was not detected in leaflet midribs of single accessions of M. lucida and M. omphalocarpa. The species identification of the CLas-positive accessions was further confirmed using all the molecular taxonomic markers consisting of the six fragments of the maternally inherited chloroplast genome and part of the nuclear-encoded internal transcribed spacer (ITS) region. The results indicated that natural infection of M. paniculata and M. sumatrana with CLas can occur in Java. To our knowledge, this is the first demonstration of the natural infection of M. sumatrana with CLas. Further studies are required to determine whether infections persist in the absence of D. citri.

Hiding in Plain Sight: A Widespread Native Perennial Harbors Diverse Haplotypes of 'Candidatus Liberibacter solanacearum' and Its Potato Psyllid Vector.

The unculturable bacterium 'Candidatus Liberibacter solanacearum' (CLso) is responsible for a growing number of emerging crop diseases. However, we know little about the diversity and ecology of CLso and its psyllid vectors outside of agricultural systems, which limits our ability to manage crop disease and understand the impacts this pathogen may have on wild plants in natural ecosystems. In North America, CLso is transmitted to crops by the native potato psyllid (Bactericera cockerelli). However, the geographic and host plant range of the potato psyllid and CLso beyond the borders of agriculture are not well understood. A recent study of historic herbarium specimens revealed that a unique haplotype of CLso was present infecting populations of the native perennial Solanum umbelliferum in California decades before CLso was first detected in crops. We hypothesized that this haplotype and other potentially novel CLso variants are still present in S. umbelliferum populations. To test this, we surveyed populations of S. umbelliferum in Southern California for CLso and potato psyllid vectors. We found multiple haplotypes of CLso and the potato psyllid associated with these populations, with none of these genetic variants having been previously reported in California crops. These results suggest that CLso and its psyllid vectors are much more widespread and diverse in North American natural plant communities than suggested by data collected solely from crops and weeds in agricultural fields. Further characterization of these apparently asymptomatic haplotypes will facilitate comparison with disease-causing variants and provide insights into the continued emergence and spread of CLso.

Diversity of 'Candidatus Liberibacter asiaticus' Strains in Texas Revealed by Prophage Sequence Analyses.

Prophages/phages are important components of the genome of 'Candidatus Liberibacter asiaticus' (CLas), an unculturable alphaproteobacterium associated with citrus huanglongbing (HLB) disease. Phage variations have significant contributions to CLas strain diversity research, which provide critical information for HLB management. In this study, prophage variations among selected CLas strains from southern Texas were studied. The CLas strains were collected from three different CLas inhabitant environments: citrus leaf, citrus root, and Asian citrus psyllid (ACP), the vector of CLas. Regardless of the different habitats and time span, more than 80% of CLas strains consistently had both Type 1 and Type 2 prophages, the same prophage type profile as in CLas strains from Florida but different to those reported in California and China. Further studies were performed on prophage type diversity. Analyses on Type 1-specific PCR amplicon sequences (encoding an endolysin protein) revealed the presence of two groups: Type 1-A, clustered around prophage SC1 originating from Florida, and Type 1-B, clustered with prophage P-SGCA5-1 originating in California. Type 1-B strains were mostly from ACP of nearby citrus orchards. On the other hand, analyses on Type 2-specific PCR amplicon sequences (encoding a putative hypothetical protein) showed a single group clustering around prophage SC2 originated from Florida, although a different Type 2 prophage has been reported in California. The presence of two distinct Type 1 prophage groups suggested the possibility of two different CLas introductions in southern Texas. The results from this study provide an initial baseline of information on genomic and population diversity of CLas in Texas.

An Inhibitor-Monitorable Single-Tube Duplex Quantitative Real-Time PCR Assay for the Detection of 'Candidatus Liberibacter asiaticus'.

Huanglongbing (HLB) is a citrus infectious disease caused by 'Candidatus Liberibacter' spp. Recently, it has begun to spread rapidly worldwide, causing significant losses to the citrus industry. Early diagnosis of HLB relies on quantitative real-time PCR assays. However, the PCR inhibitors found in the nucleic acid extracted from plant materials pose challenges for PCR assays because they may result in false-negative results. Internal standard (IS) can be introduced to establish a single-tube duplex PCR for monitoring the influence of the PCR inhibitor, but it also brings the risk of false-negative results because the amplification of IS may compete with the target. To solve this problem, we proposed a mutation-enhanced single-tube duplex PCR (mSTD-PCR) containing IS with mutant-type primers. By introducing the 3'-terminal mutation in the primer of IS to weaken its amplification reaction and its inhibition of 'Candidatus Liberibacter asiaticus' (CLas) detection, the sensitivity and quantitative accuracy of CLas detection will not be affected by IS. In evaluating the sensitivity of CLas detection using simulation samples, the mSTD-PCR showed consistent sensitivity at 25 copies per test compared with the single-plex CLas assay. The detection result of 30 leaves and 30 root samples showed that the mSTD-PCR could recognize false-negative results caused by the PCR inhibitors and reduce workload by 48% compared with the single-plex CLas assay. Generally, the proposed mSTD-PCR provides a reliable, efficient, inhibitor-monitorable, quantitative screening method for accurately controlling HLB and a universal method for establishing a PCR assay for various pathogens.

In situ diagnosis of mature HLB-asymptomatic citrus fruits by laser-induced breakdown spectroscopy.

Laser-induced breakdown spectroscopy (LIBS) is a promising alternative to conventional methods in classifying citrus huanglongbing (HLB). Mature citrus fruits with similar features were picked and divided into healthy and HLB-asymptomatic groups. LIBS spectra and images were collected by focusing a laser on fresh fruit surfaces without sample preparation. The pH value and soluble solids content of juice as the indicators of acidity and sugar were detected, and the content of Ca, Zn, and K in peel and pulp was analyzed. The characteristic lines from LIBS spectra were extracted by continuous wavelet transform and principal component analysis (PCA). The t-test of these indicators displayed significant difference between the two groups. Fisher discriminant analysis and multilayer perception neural network (MLP) were applied to identify the disease. The classification accuracy reached 100% by PCA-MLP. The results show that LIBS can realize in situ detection of citrus HLB fruits.

Diverse symbiovars nodulating cowpea (Vigna unguiculata L. Walp.) in highly adaptable agro-ecological zones in Mozambique.

The presence of effective microsymbionts in the soil and their compatibility with the host plant are the key determinants to the N2 fixation process. In Sub-Saharan Africa, nitrogen fixation in locally adapted cowpea and the distribution of their symbiovars are not well understood. The Aim of the study was to assess the distribution and symbiotic phylogenetic position of cowpea microsymbionts. Root nodules were sampled from various cowpea genotypes planted in Agro-Ecological Zone 7 and 8 (AEZ 7 and AEZ 8). Root-nodule bacteria were isolated and their molecular characterization was conducted. Physicochemical properties of soil were recorded. Enterobacterial Repetitive Intergenic Consensus (ERIC) distribution patterns in rhizobial genomes resulted in genetically diverse rhizobial population in Northern Mozambique. Principal component analysis showed that location-specific soil environment determined the presence of particular microsymbionts. Based on 16S rRNA and symbiotic gene analysis many diverse symbiovars were found in Mozambican soils. With few discrepancies, the results further confirmed the coevolution of the nifH, nodD, nodC and nodY/K genes, which was indicative of natural events such as vertical/horizontal gene transfer. The results suggested that ecological and phylogenetic studies of the microsymbionts are necessary to better reflect symbiovar identification and the ecological adaptation of the cowpea-nodulating rhizobial community.

Comprehensive characterization of stress tolerant bacteria with plant growth-promoting potential isolated from glyphosate-treated environment.

The application of plant growth-promoting bacteria in agricultural systems is an efficient and environment-friendly strategy to improve crop yields and maintain soil quality. However, as different soils have diverse and specific ecological characteristics and may represent adverse abiotic conditions, in vivo application requires the careful selection of the desired beneficial microorganisms. In this study we report Ensifer adhaerens SZMC 25856 and Pseudomonas resinovorans SZMC 25875 isolates recovered from glyphosate-treated soil to possess yet undiscovered plant growth-enhancing potential. The strains were found to promote the growth of tomato seedlings significantly, to have the ability of synthesizing indole-3-acetic acid and siderophores, to tolerate pH in the range of 6.59-7.96, salinity up to 12.5 g L-1 NaCl and drought up to 125 g L-1 polyethylene glycol 6000, as well as to survive in the presence of various pesticides including glyphosate, diuron, chlorotoluron, carbendazim and thiabendazole, and heavy metals such as Al, Fe, Mn, Zn, Pb and Cu. The plant growth-promoting traits of the examined E. adhaerens and P. resinovorans isolates and their tolerance to numerous abiotic stress factors make them promising candidates for application in different agricultural environments, including soils polluted with glyphosate.

Isolation and characterization of bacteria from activated sludge capable of degrading 17α-ethinylestradiol, a contaminant of high environmental concern.

The compound 17α-ethinylestradiol (EE2) is a synthetic oestrogen which is classified as a group 1 carcinogen by the World Health Organization. Together with other endocrine disruptor compounds, EE2 has been included in the surface water Watch List by the European Commission, since it causes severe adverse effects in ecosystems. Thus, it became a high priority to find or improve processes such as biodegradation of EE2 to completely remove this drug from the wastewater treatment plants (WWTPs). The present study aimed at the isolation of bacteria capable of degrading EE2 using environmental samples, namely a sludge from the Faro Northwest WWTP. Four isolates with ability to grow in the presence of 50 mg l-1 EE2 were obtained. The analysis of 16SrRNA gene sequences identified the isolated bacteria as Acinetobacter bouvetii, Acinetobacter kookii, Pantoea agglomerans and Shinella zoogloeoides. The results of biodegradation assays showed that Acinetobacter bouvetii, Acinetobacter kookii, Pantoea agglomerans and Shinella zoogloeoides were able to degrade 47±4 %, 55±3 %, 64±4% and 35±4 %, respectively of 13 mg l-1 EE2 after 168 h at 28 °C. To the best of our knowledge, these bacterial isolates were identified as EE2 degraders for the first time. In a preliminary experiment on the identification of metabolic products resulting from EE2 degradation products such as estrone (E1), γ-lactone compounds, 2-pentanedioic acid and 2-butenedioic acid an intermediate metabolite of the TCA cycle, were detected.

ACC deaminase-producing Ensifer adhaerens KS23 enhances proximate nutrient of Pisum sativum L. cultivated in high altitude.

A phytohormone producing, N2-fixing and 1-aminocyclopropane-1-carboxylate (ACC) deaminase synthesizing bacterium Ensifer adhaerens KS23 effectively increased the yield and nutritional contents of Pisum sativum var. Arkel. The isolate KS23 showed positive ACC deaminase activity with 174.2 (nmol of α-ketobutyrate/g-1 biomass½ h-1) a 9.7-fold increase in glutathione S-transferase activity. The proximate analysis exhibited an increased yield of protein (21.45%), carbohydrate (38.90%), sulphur (29.94%) starch (27.52%), total ash (35.57%), fat content (27.5%), nitrogen (24.06%) and hydrogen (17.91%) in treated seeds of P. sativum as compared to untreated crop seeds in field trials at Srikot village, Srinagar-246,174 (Garhwal) India. The most desirable essential and non-essential amino-acids content was also enhanced simultaneously by E. adhaerens KS23 as compared to non-treated crop seeds. This study revealed the enhancement of various nutritional contents resulting in quality improvement and an increase in growth productivity of pea. This study provides an encouraging result that may benefit the marginal income of farmers belonging mainly to hilly regions who are dependent on traditional methods of farming and thus improving their economy.

Isolation of a member of the candidate phylum 'Atribacteria' reveals a unique cell membrane structure.

A key feature that differentiates prokaryotic cells from eukaryotes is the absence of an intracellular membrane surrounding the chromosomal DNA. Here, we isolate a member of the ubiquitous, yet-to-be-cultivated phylum 'Candidatus Atribacteria' (also known as OP9) that has an intracytoplasmic membrane apparently surrounding the nucleoid. The isolate, RT761, is a subsurface-derived anaerobic bacterium that appears to have three lipid membrane-like layers, as shown by cryo-electron tomography. Our observations are consistent with a classical gram-negative structure with an additional intracytoplasmic membrane. However, further studies are needed to provide conclusive evidence for this unique intracellular structure. The RT761 genome encodes proteins with features that might be related to the complex cellular structure, including: N-terminal extensions in proteins involved in important processes (such as cell-division protein FtsZ); one of the highest percentages of transmembrane proteins among gram-negative bacteria; and predicted Sec-secreted proteins with unique signal peptides. Physiologically, RT761 primarily produces hydrogen for electron disposal during sugar degradation, and co-cultivation with a hydrogen-scavenging methanogen improves growth. We propose RT761 as a new species, Atribacter laminatus gen. nov. sp. nov. and a new phylum, Atribacterota phy. nov.

Root-associated endophytic bacterial community composition and structure of three medicinal licorices and their changes with the growing year.

BACKGROUND: The dried roots and rhizomes of medicinal licorices are widely used worldwide as a traditional medicinal herb, which are mainly attributed to a variety of bioactive compounds that can be extracted from licorice root. Endophytes and plants form a symbiotic relationship, which is an important source of host secondary metabolites. RESULTS: In this study, we used high-throughput sequencing technology and high-performance liquid chromatography to explore the composition and structure of the endophytic bacterial community and the content of bioactive compounds (glycyrrhizic acid, liquiritin and total flavonoids) in different species of medicinal licorices (Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata) and in different planting years (1-3 years). Our results showed that the contents of the bioactive compounds in the roots of medicinal licorices were not affected by the species, but were significantly affected by the main effect growing year (1-3) (P < 0.05), and with a trend of stable increase in the contents observed with each growing year. In 27 samples, a total of 1,979,531 effective sequences were obtained after quality control, and 2432 effective operational taxonomic units (OTUs) were obtained at 97% identity. The phylum Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes, and the genera unified-Rhizobiaceae, Pseudomonas, Novosphingobium, and Pantoea were significantly dominant in the 27 samples. Distance-based redundancy analysis (db-RDA) showed that the content of total flavonoids explained the differences in composition and distribution of endophytic bacterial communities in roots of cultivated medicinal liquorices to the greatest extent. Total soil salt was the most important factor that significantly affected the endophytic bacterial community in soil factors, followed by ammonium nitrogen and nitrate nitrogen. Among the leaf nutrition factors, leaf water content had the most significant effect on the endophytic bacterial community, followed by total phosphorus and total potassium. CONCLUSIONS: This study not only provides information on the composition and distribution of endophytic bacteria in the roots of medicinal licorices, but also reveals the influence of abiotic factors on the community of endophytic bacteria and bioactive compounds, which provides a reference for improving the quality of licorice.

Detoxification and bioaugmentation potential for acetaminophen and its derivatives using Ensifer sp. isolated from activated sludge.

Acetaminophen (APAP), a widely used analgesic-antipyretic drug, is frequently detected in the environment and may pose ecological risks to aquatic communities. In this work, an APAP-degrading organism, designated as Ensifer sp. POKHU, was isolated from activated sludge (AS) enriched with APAP. POKHU degraded up to 630 mg/L of APAP without substrate inhibition. The bacterium metabolized APAP to hydroquinone (HQ) via 4-aminophenol (4-AP). APAP derivatives, 4AP, HQ, and 1,4-benzoquinone (BQ), frequently detected in the environment, were found to inhibit nitrogen metabolism (ammonium oxidation) to a greater extent than APAP. POKHU had the ability to degrade varying levels (0.4-40 mg/L) of 4-AP, HQ, and BQ, which indicated a great potential for detoxification in environments contaminated with both APAP and its derivatives. The addition of POKHU to fresh AS samples taken from a wastewater treatment plant greatly increased the biotransformation rates of APAP from 5.6 d-1 (no POKHU augmentation) to >20.0 d-1 (5% POKHU). Bioaugmentation with POKHU reduced 400 µg/L of APAP to levels below its ecotoxicity threshold within 4 h, which is shorter than the typical hydraulic retention times for full-scale AS processing. Overall, this study identified a new auxiliary biological agent for APAP detoxification, which could degrade both APAP and its metabolic derivatives (those that can be more toxic than the parent contaminant, APAP). The results have practical implications for developing a biological means (detoxification and bioaugmentation) of treating high-strength pharmaceutical waste streams, such as wastewater from hospitals and drug manufactures, and of landfill leachates.

First Report of HLB Causal Agent in Psyllid in State of Bahia, Brazil.

The state of Bahia ranks fourth in the national rank for citrus production, and the region of Chapada Diamantina is emerging an important producer of orange for fresh fruit market. Huanglongbing (HLB) is the major phytosanitary threat to Bahia citriculture. In Brazil, the disease was first reported in 2004 in São Paulo state. The bacterium Candidatus Liberibacter asiaticus (CLas) is one of the causal agents of HLB, which is transmitted by the insect vector Diaphorina citri Kuwayama (Hemiptera: Liviidae). Bahia is a HLB-free area; therefore, it is essential to monitor its citrus-producing areas to early detect any possible introduction of the CLas. This study aimed to monitor the presence of the bacteria in the insect vector. Diaphorina citri samples were collected from 2011 to 2014 in different cities located at Chapada Diamantina region and tested by qPCR for the presence of CLas. Three samples were considered positive to bacterium, and all from psyllids collected on Murraya paniculata in the city of Seabra.

Biochemical, functional and molecular characterization of pigeon pea rhizobia isolated from semi-arid regions of India.

Pigeon pea (Cajanus cajan (L.) Millspaugh) is among the top ten legumes grown globally not only having high tolerance to environmental stresses along, but also has the high biomass and productivity with optimal nutritional profiles. In the present study, 55 isolates of rhizobia were identified from 22 nodule samples of pigeon pea collected from semi-arid regions of India on the basis of morphological, biochemical, plant growth promoting activities and their ability to tolerate the stress conditions viz. pH, salt, temperature and drought stress. Amongst all the 55 isolates, 37 isolates showed effective nodulation under in vitro conditions in pigeon pea. Further, five isolates having multiple PGP activities and high in vitro symbiotic efficiency were subjected to 16S rRNA sequencing and confirmed their identities as Rhizobium, Mesorhizobium, Sinorhizobium sp. Further these 37 isolates were characterized at molecular level using ARDRA and revealed significant molecular diversity. Based on UPGMA clustering analysis, these isolates showed significant molecular diversity. The high degree of molecular diversity is due to mixed cropping of legumes in the region. The assessment of genetic diversity and molecular characterization of novel strains is a very important tool for the replacement of ineffective rhizobial strains with the efficient strains for the improvement in the nodulation and pigeon pea quality. The pigeon pea isolates with multiple PGPR activities could be further used for commercial production.

Ciceribacter ferrooxidans sp. nov., a nitrate-reducing Fe(II)-oxidizing bacterium isolated from ferrous ion-rich sediment.

A nitrate-reducing Fe(II)-oxidizing bacterial strain, F8825T, was isolated from the Fe(II)-rich sediment of an urban creek in Pearl River Delta, China. The strain was Gram-negative, facultative chemolithotrophic, facultative anaerobic, non-spore-forming, and rod-shaped with a single flagellum. Phy-logenetic analysis based on 16S rRNA gene sequencing indicated that it belongs to the genus Ciceribacter and is most closely related to C. lividus MSSRFBL1T (99.4%), followed by C. thiooxidans F43bT (98.8%) and C. azotifigens A.slu09T (98.0%). Fatty acid, polar lipid, respiratory quinone, and DNA G + C content analyses supported its classification in the genus Ciceribacter. Multilocus sequence analysis of concatenated 16S rRNA, atpD, glnII, gyrB, recA, and thrC suggested that the isolate was a novel species. DNA-DNA hybridization and genome sequence comparisons (90.88 and 89.86%, for values of ANIm and ANIb between strains F8825T with MSSRFBL1T, respectively) confirmed that strain F8825T was a novel species, different from C. lividus MSSRFBL1T, C. thiooxidans F43bT, and C. azotifigens A.slu09T. The physiological and biochemical properties of the strain, such as carbon source utilization, nitrate reduction, and ferrous ion oxidation, further supported that this is a novel species. Based on the polyphasic taxonomic results, strain F8825T was identified as a novel species in the genus Ciceribacter, for which the name Ciceribacter ferrooxidans sp. nov. is proposed. The type strain is F8825T (= CCTCC AB 2018196T = KCTC 62948T).

Canine olfactory detection of a vectored phytobacterial pathogen, Liberibacter asiaticus, and integration with disease control.

Early detection and rapid response are crucial to avoid severe epidemics of exotic pathogens. However, most detection methods (molecular, serological, chemical) are logistically limited for large-scale survey of outbreaks due to intrinsic sampling issues and laboratory throughput. Evaluation of 10 canines trained for detection of a severe exotic phytobacterial arboreal pathogen, Candidatus Liberibacter asiaticus (CLas), demonstrated 0.9905 accuracy, 0.8579 sensitivity, and 0.9961 specificity. In a longitudinal study, cryptic CLas infections that remained subclinical visually were detected within 2 wk postinfection compared with 1 to 32 mo for qPCR. When allowed to interrogate a diverse range of in vivo pathogens infecting an international citrus pathogen collection, canines only reacted to Liberibacter pathogens of citrus and not to other bacterial, viral, or spiroplasma pathogens. Canines trained to detect CLas-infected citrus also alerted on CLas-infected tobacco and periwinkle, CLas-bearing psyllid insect vectors, and CLas cocultured with other bacteria but at CLas titers below the level of molecular detection. All of these observations suggest that canines can detect CLas directly rather than only host volatiles produced by the infection. Detection in orchards and residential properties was real time, ∼2 s per tree. Spatiotemporal epidemic simulations demonstrated that control of pathogen prevalence was possible and economically sustainable when canine detection was followed by intervention (i.e., culling infected individuals), whereas current methods of molecular (qPCR) and visual detection failed to contribute to the suppression of an exponential trajectory of infection.

An In Vitro Pipeline for Screening and Selection of Citrus-Associated Microbiota with Potential Anti-"Candidatus Liberibacter asiaticus" Properties.

Huanglongbing (HLB) is a destructive citrus disease that is lethal to all commercial citrus plants, making it the most serious citrus disease and one of the most serious plant diseases. Because of the severity of HLB and the paucity of effective control measures, we structured this study to encompass the entirety of the citrus microbiome and the chemistries associated with that microbial community. We describe the spatial niche diversity of bacteria and fungi associated with citrus roots, stems, and leaves using traditional microbial culturing integrated with culture-independent methods. Using the culturable sector of the citrus microbiome, we created a microbial repository using a high-throughput bulk culturing and microbial identification pipeline. We integrated an in vitro agar diffusion inhibition bioassay into our culturing pipeline that queried the repository for antimicrobial activity against Liberibacter crescens, a culturable surrogate for the nonculturable "Candidatus Liberibacter asiaticus" bacterium associated with HLB. We identified microbes with robust inhibitory activity against L. crescens that include the fungi Cladosporium cladosporioides and Epicoccum nigrum and bacterial species of Pantoea, Bacillus, and Curtobacterium Purified bioactive natural products with anti-"Ca. Liberibacter asiaticus" activity were identified from the fungus C. cladosporioides Bioassay-guided fractionation of an organic extract of C. cladosporioides yielded the natural products cladosporols A, C, and D as the active agents against L. crescens This work serves as a foundation for unraveling the complex chemistries associated with the citrus microbiome to begin to understand the functional roles of members of the microbiome, with the long-term goal of developing anti-"Ca Liberibacter asiaticus" bioinoculants that thrive in the citrus holosystem.IMPORTANCE Globally, citrus is threatened by huanglongbing (HLB), and the lack of effective control measures is a major concern of farmers, markets, and consumers. There is compelling evidence that plant health is a function of the activities of the plant's associated microbiome. Using Liberibacter crescens, a culturable surrogate for the unculturable HLB-associated bacterium "Candidatus Liberibacter asiaticus," we tested the hypothesis that members of the citrus microbiome produce potential anti-"Ca Liberibacter asiaticus" natural products with potential anti-"Ca Liberibacter asiaticus" activity. A subset of isolates obtained from the microbiome inhibited L. crescens growth in an agar diffusion inhibition assay. Further fractionation experiments linked the inhibitory activity of the fungus Cladosporium cladosporioides to the fungus-produced natural products cladosporols A, C, and D, demonstrating dose-dependent antagonism to L. crescens.

Beneficial bacteria activate nutrients and promote wheat growth under conditions of reduced fertilizer application.

BACKGROUND: Excessive application of chemical fertilizer has exerted a great threat to soil quality and the environment. The inoculation of plants with plant-growth-promoting rhizobacteria (PGPR) has emerged as a great prospect for ecosystem recovery. The aim of this work to isolate PGPRs and highlights the effect of bacterial inoculants on available N/P/K content in soil and on the growth of wheat under conditions of reduced fertilizer application. RESULTS: Thirty-nine PGPRs were isolated and tested for their growth-promoting potential. Thirteen isolates had nitrogen fixation ability, of which N9 (Azotobacter chroococcum) had the highest acetylene reduction activity of 156.26 nmol/gh. Eleven isolates had efficient phosphate solubilizing ability, of which P5 (Klebsiella variicola) released the most available phosphorus in liquid medium (231.68 mg/L). Fifteen isolates had efficient potassium solubilizing ability, of which K13 (Rhizobium larrymoorei) released the most available potassium in liquid medium (224.66 mg/L). In culture medium supplemented with tryptophan, P9 (Klebsiella pneumoniae) produced the greatest amount of IAA. Inoculation with the bacterial combination K14 + 176 + P9 + N8 + P5 increased the alkali-hydrolysed nitrogen, available phosphorus and available potassium in the soil by 49.46, 99.51 and 19.38%, respectively, and enhanced the N, P, and K content of wheat by 97.7, 96.4 and 42.1%, respectively. Moreover, reducing fertilizer application by 25% did not decrease the available nitrogen, phosphorus, and potassium in the soil and N/P/K content, plant height, and dry weight of wheat. CONCLUSIONS: The bacterial combination K14 + 176 + P9 + N8 + P5 is superior candidates for biofertilizers that may reduce chemical fertilizer application without influencing the normal growth of wheat.

Quenching autofluorescence in the alimentary canal tissues of Bactericera cockerelli (Hemiptera: Triozidae) for immunofluorescence labeling.

Immunofluorescence has been widely used to localize microbes or specific molecules in insect tissues or cells. However, significant autofluorescence is frequently observed in tissues which can interfere with the fluorescent identification of target antigens, leading to inaccurate or even false positive fluorescent labeling. The alimentary canal of the potato psyllid, Bactericera cockerelli Sulc, exhibits intense autofluorescence, hindering the application of immunolocalization for the detection and localization of the economically important pathogen transmitted by this insect, "Candidatus Liberibacter solanacearum" (Lso). In the present study, we tested the use of irradiation, hydrogen peroxide (H2 O2 ) and Sudan black B (SBB) treatments to reduce the autofluorescence in the B. cockerelli alimentary canal tissues. Furthermore, we assessed the compatibility of the above-mentioned treatments with Lso immunolocalization and actin staining using phalloidin. Our results showed that the autofluorescence in the alimentary canal was reduced by irradiation, H2 O2 , or SBB treatments. The compatibility assays indicated that irradiation and H2 O2 treatment both greatly reduced the fluorescent signal associated with Lso and actin. However, the SBB incubation preserved those target signals, while efficiently eliminating autofluorescence in the psyllid alimentary canal. Therefore, herein we propose a robust method for reducing the autofluorescence in the B. cockerelli alimentary canal with SBB treatment, which may improve the use of immunofluorescence labeling in this organism. This method may also have a wide range of uses by reducing the autofluorescence in other arthropod species.