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A previous study found that a biocontrol bacterium, Bacillus pumilus HR10, inhibited the Sphaeropsis shoot blight disease of pine, and the fermentation broth of HR10 strain contained protein antifungal substances. The optimal formulation of the fermentation medium for the antagonistic substance of B. pumilus HR10 was finally obtained by single-factor test, Packett-Burman test, steepest ascent test and Box-Behnken Design (BBD) response surface test, and the best formulation of the fermentation medium for the antagonistic substance of B. pumilus HR10 was 12 g/L corn meal, 15 g/L beef extract and 13 g/L magnesium sulfate, with a predicted bacterial inhibition rate of 89%. The fermentation filtrate of B. pumilus HR10 cultured with the optimized medium formulation was verified to have an inhibition rate of (87.04 ± 3.2) % on the growth of Sphaeropsis sapinea by three replicate tests. The antagonistic crude protein of B. pumilus HR10 were further isolated and identified using HiTrap Capto Q strong Ion-Exchange Chromatography and LC–MS-MS, and it was speculated that glycoside hydrolase (Ghy), beta-glucanase (Beta), arabinogalactan endonuclease β-1,4-galactanase (Arab), and immunosuppressant A (ImA) are proteins with antagonistic activity against S. sapinea in the B. pumilus HR10. Keypoints: Fermentation medium with high antagonistic activity of HR10 strain was determined. Antagonistic crude protein of HR10 strain purified by Ion-Exchange Chromatography. Composition of the antagonist protein of HR10 strain was analyzed using LC-MS-MS. [ABSTRACT FROM AUTHOR]

Cerebral cortical vein thrombosis (CCVT) is a rare type of cerebral venous thrombosis, which is frequently combined with cerebral venous sinus thrombosis (CVST). We aimed to compare the difference of clinical features between the isolated and the combined subtypes of CCVT. A literature search was conducted utilizing the PubMed Central and EMBASE databases to identify studies up to Dec 2019. Clinical manifestations, presumable risk factors, imaging modalities, radiological findings, treatment, and prognosis in patients with CCVT were recorded. 335 publications were identified (n = 325, 141 males and 184 females, mean age 40.24 ± 16.26 years). Headaches (46.8%), motor/sensory disorders (43.3%), and seizures (42.5%) were commonly seen. Pregnancy/postpartum (n = 29), oral contraception use (n = 15), fertility drug use (n = 4) ranked the top three comorbidities of CCVT in female patients, while for general populations, thrombophilia, invasive interventions in the cerebrospinal system, as well as malignancy, would be the common risk factors. MRV and DSA were more likely to confirm diagnosis. More than 30% of CCVT presented brain lesions, including infarction (6.5%) and hemorrhage (24.0%). Isolated CCVT was prone to develop hemorrhagic infarction while combined CCVT was more likely to have ischemic lesions. More than 90% of the patients acquired good outcomes at discharge or short-term follow-up (within one year). There is a difference between Isolated CCVT and CCVT combined CVST on the sites and types of brain lesions. MRV and DSA may contribute to the final diagnosis. Most patients acquired complete or partial recovery of clinical symptoms or imaging presentations after long-term anticoagulation (3–6 months). [ABSTRACT FROM AUTHOR]

Many studies have focused on the significant role of biofilm formation by Bacillus in the biocontrol process. Bacillus pumilus HR10 is a plant growth-promoting rhizobacterium with multiple biocontrol functions, including promoting growth, controlling pathogens, and assisting in the formation of mycorrhizae. Currently, there is no relevant report on the biofilm formation of B. pumilus HR10 and its influencing factors. B. pumilus HR10 was found to easily form a stable biofilm structure on the surface of media, with awesome swarming ability. The optimum temperature for biofilm formation was 37 °C. B. pumilus HR10 formed most obviously at pH 7.0 and was not extremely sensitive to acidic or alkaline conditions. Most of the polysaccharide components of plant root exudates promoted the biofilm formation by B. pumilus HR10, with glucose having the greatest promoting effect. Low concentrations of Fe2+, Mg2+, Ca2+, K+, and Na+ enhanced biofilm formation. In summary, biofilm formation can improve the tolerance of B. pumilus HR10 to salt and certain heavy metal ion stresses and contribute to its application in different plants and soils with high salinity or heavy metals in the field. [ABSTRACT FROM AUTHOR]

Bursaphelenchus xylophilus is an economically important pathogen that has caused serious damage to pine forests. Fatty acid and retinol-binding proteins (FARs) are nematode-specific proteins. One FAR gene, Bx-FAR-1, was identified from the transcriptome of B. xylophilus as a putative effector. It was upregulated in both highly and weakly virulent isolates in early infection stages. Transient expression assays showed that Bx-FAR-1 inhibited BAX- and INF1-induced cell death in Nicotiana benthamiana when secreted into the intracellular site. Bx-FAR-1 was expressed in the glandular tissues, intestines, and seminal vesicles of nematodes by in situ hybridization. Silencing Bx-FAR-1 via in vitro RNA interference attenuated the reproductive ability and pathogenicity of B. xylophilus as well as increased the expression of the pathogenesis-related gene 6 and JA biosynthesis gene LOX-5 (lipoxygenase-5) in pine trees. These results suggest that Bx-FAR-1 suppresses the plant immune response in the N. benthamiana intracellular site and may contribute to the pathogenicity of B. xylophilus in the early infection stages. [ABSTRACT FROM AUTHOR]

As important plant growth-promoting rhizobacteria, phosphate-solubilizing bacteria (PSB) fix nitrogen, dissolve potassium, promote growth, improve the soil micro-environment, and enhance soil fertility. A high-efficiency PSB strain from the pine tree rhizosphere, Burkholderia multivorans WS-FJ9, was screened in our laboratory. In this study, using a Bio Screener fully automatic microbial growth curve meter to determine the growth of the WS-FJ9 strain in phosphate-removing medium, the growth and mineral phosphate solubilization of WS-FJ9 were measured by Mo-Sb colorimetry and organophosphate-solubilization plate assays. Second-generation sequencing technology was used to obtain genomic information and to analyze possible phosphate decomposition genes. The related expression levels of these genes under different soluble phosphate levels were determined by quantitative real-time PCR. The results showed that WS-FJ9 had strong adaptability and capacity for mineral phosphate solubilization at low soluble phosphate levels, which is characterized by its low soluble phosphate induction and high soluble phosphate inhibition. The amount of solubilized mineral phosphate could exceed 140 mg/L. The total length of the WS-FJ9 genome was 7,497,552 bp after splicing, and the GC content was 67.37%. Eight phosphate-related genes were selected to determine their expression patterns at different soluble phosphate levels. Among them, AP-2, GspE and GspF were only related to organic phosphate, HlyB was only related to inorganic phosphate, and PhoR, PhoA, AP-1 and AP-3 were related to both. The WS-FJ9 strain utilizes multiple pathways for mineral phosphate solubilization, and the solubilization processes of different phosphate sources are interrelated and independent, indicating that the WS-FJ9 strain can adapt to different phosphate source environments and has good potential for future applications. [ABSTRACT FROM AUTHOR]

With the increasing popularity of urban landscaping, there is a greater need to address iron deficiency and chlorosis in Cinnamomum camphora. Beneficial microorganisms can play an important role in improving plant iron nutrition. In this study, we evaluated the greening effect that results from inoculating C. camphora with Rahnella aquatilis JZ-GX1. We conducted in vitro experiments to determine the effectiveness of JZ-GX1 in reducing extracellular iron(III) ions in siderophores and producing organic acids under low-iron conditions. Potted, chlorotic C. camphora seedlings were inoculated, and parameters related to growth and iron nutritional status were measured. Inoculation by JZ-GX1 had a more pronounced greening effect on C. camphora than the traditional iron sulfate treatment for plant iron deficiency. Inoculated plants exhibited higher levels of active iron in the leaves, enhanced rhizosphere acidification capacity and ferric chelate reductase activity. Furthermore, the complete root system and chloroplast developmental structures were observed. Our results demonstrate the potential roles of R. aquatilis JZ-GX1 in promoting iron absorption in C. camphora. [ABSTRACT FROM AUTHOR]

A salt-tolerant microbe strain JYZ-SD2 was investigated to develop biological soil amendments to stimulate salix growth and acclimation in costal salt-affected soils. The salt tolerance mechanism of strain JYZ-SD2 was investigated by detecting the salt-tolerant growth characteristics, biofilm formation, ion distribution, secondary metabolites, and zymogram profiling. The strain was identified by physiological and biochemical characteristics (Biolog), 16S rDNA sequencing, and cry1/7/9 gene expressing. With increasing of NaCl concentration, strain JYZ-SD2 adapted to the increased osmotic pressure by prolonging the retardation period, slowing down the growth rate of the logarithmic phase, increasing spo0A gene expression, increasing biofilm formation, reducing Na+ uptake, and changing the expression of metabolites and intracellular soluble proteins. The results showed that strain JYZ-SD2 could be assigned to Bacillus cereus. [ABSTRACT FROM AUTHOR]

In recent years, crown gall of flowering cherry saplings, caused by Agrobacterium tumefaciens, become a serious disease in Xuzhou, China. In this study, the average disease incidence rate in the flowering cherry sapling was 76%, and the disease index was 38. Three-year-old flowering cherry saplings were the most susceptible, and the low-lying terrain was the most favorable condition for the disease. Two strains with distinct antagonistic effects against Agrobacterium tumefaciens were screened simultaneously from the rhizospheric soil of flowering cherry saplings and were named JK-XZ3 and JK-XZ8. The JK-XZ3 and JK-XZ8 strains were identified as Bacillus albus and B. velezensis, respectively, via morphological observation, physiological analysis, biochemical identification and multilocus sequence analysis. The strains JK-XZ3 and JK-XZ8 could effectively prevent the growth of A. tumefaciens. In a greenhouse experiment, the incidence of crown gall in flowering cherry seedlings without the JK-XZ3 and JK-XZ8 treatments was 100%, and the mean diameter of the root nodules was 18.8 mm. The disease incidence in seedlings treated with the JK-XZ3 and JK-XZ8 strains was 16.7% and 22.3%, respectively, and the mean diameter of the root nodules was 6.5 mm and 8.4 mm, respectively. In the field trials, the incidence of crown gall in untreated cherry saplings was 86%, while the incidence of crown gall in saplings treated with the JK-XZ3 and JK-XZ8 strains was 32% and 38%, respectively. These results indicate that the JK-XZ3 and JK-XZ8 strains have a high potential as biological control agents for crown gall of flowering cherry saplings. [ABSTRACT FROM AUTHOR]

The genus Colletotrichum includes a number of important plant pathogens, which cause anthracnose diseases on a broad range of hosts in the world. In recent years, walnut has been severely damaged by anthracnose disease in China with significant yield losses. Thus, it is necessary to verify the etiology of anthracnose on walnut using both morphological and molecular approaches. In 2014, walnut fruits with anthracnose symptoms were collected from five walnut orchards in Shandong Province, China, and 24 isolates were isolated. Among them, six similar single-spore isolates obtained were used for pathogenicity testing of walnut anthracnose. Acervuli were brown, circular and the average size was 50.4-101.8 μm. Conidiophores were hyaline, septate, not branched or branching at the base, conidiogenous cells were enteroblastic, phialidic, hyaline, cylindrical, ampulliform. Conidia were single celled, smooth-walled with a large guttule, colorless, fusiform to cylindrical, and had obtuse to slightly rounded ends. The size of conidia was (11.6-)13-15(−16.2) × (4.1-)4.6-5.6(−6) μm. Appressoria were brown, ovoid to ellipsoid or slightly irregularly to irregularly shaped, and the average size was 6.8-9 × 5.1-6.5 μm. Pathogenicity of the isolates to fruits and leaves were compared, and genes from all six isolates were sequenced. The isolates were identified as C. siamense based on four-gene phylogenetic analyses (ribosomal DNA-ITS, GAPDH, ACT and CHS-1) and morphological as well as cultural characters. This is the first report of C. siamense as a causal agent of anthracnose of walnut in China. [ABSTRACT FROM AUTHOR]

Phytate-mineralizing rhizobacteria (PMR) play an important role in providing phosphorus for the sustainable plant growth. It is important to investigate the ability of PMR to produce phytase under different phosphate levels for its application. The effects of different concentrations of soluble phosphate on the ability of phytate mineralization of Pseudomonas fluorescens JZ-DZ1, a phytate-mineralizing rhizobacterium, were investigated in both solid and liquid media. The results on solid media showed that halo zone width gradually reduced with concentrations of soluble phosphate increasing from 0.05 to 20 mM, indicating the reduction of the ability of phytate mineralization. The results were consistent with the quantitative detection of phytase activity from the overall trend. An 1866-bp β-propeller phytase (BPP) gene ( phyPf) was cloned from the strain, and the deduced amino acid sequence of phyPf shared 98 % of identity with a known BPP from Pseudomonas sp. BS10-3 (AJF36073.1). The results of relative real-time quantitative PCR assay showed that the expression of phyPf was induced by a low concentration (0.1 mM) of soluble phosphate, suggesting that BPP secretion was regulated by gene phyPf. The BPP-harboring bacterium P. fluorescens JZ-DZ1 with low phosphate-inducible ability of phytate mineralization could be potentially applied to promote phosphorus uptake for plants in the future. [ABSTRACT FROM AUTHOR]

If mycorrhizal formation could be enhanced by co-inoculation with mycorrhiza helper bacteria (MHB) which promote rapid root colonization by specific ectomycorrhizal fungi, this would be of advantage to the poplar forest industry. A number of poplar rhizobacterial strains were isolated from 11 regions of the eastern China. Four of the isolates, SY15, DZ18, HLJ4, and PY10, were characterized as MHB potential strains based on their positive effect on growth of ectomycorrhizal fungi Pisolithus tinctorius (Pers.) and Lactarius insulsus (Fr.). Under greenhouse conditions, one of the bacterial isolate, DZ18, significantly promoted the poplar trees growth and ectomycorrhizal colonization of P. tinctorius and L. insulsus on Populus deltoides Marsh. In contrast, the other three isolates SY15, HLJ4, and PY10 promoted fungal growth in vitro experiments but did not enhance ectomycorrhizal (ECM) formation in the greenhouse experiment. Therefore, it was concluded that DZ18 can be considered as an MHB strain. DZ18 was identified as Bacillus sp. based on morphological, physiological, and biochemical analyses in combination with analysis of 16S rDNA gene sequences. [ABSTRACT FROM AUTHOR]

Burkholderia cepacia complex (Bcc) is a group of bacteria with conflicting biological characteristics, which make them simultaneously beneficial and harmful to humans. They have been exploited for biocontrol, bioremediation, and plant growth promotion. However, their capacity as opportunistic bacteria that infect humans restricts their biotechnological applications. Therefore, the risks of using these bacteria should be assessed. In this study, Burkholderia multivorans WS-FJ9 originally isolated from pine rhizosphere, which was shown to be efficient in solubilizing phosphate, was evaluated with respect to its biosafety, colonization in poplar rhizosphere, and growth-promoting effects on poplar seedlings. Pathogenicity of B. multivorans WS-FJ9 on plants was determined experimentally using onion and tobacco as model plants. Onion bulb inoculated with B. multivorans WS-FJ9 showed slight hypersensitive responses around the inoculation points, but effects were not detectable based on the inner color and odor of the onion. Tobacco leaves inoculated with B. multivorans WS-FJ9 exhibited slightly water-soaked spots around the inoculation points, which did not expand or develop into lesions even with repeated incubation. Pathogenicity of the strain in alfalfa, which has been suggested as an alternative Bcc model for mice, was not detectable. Results from gene-specific polymerase chain reactions showed that the tested B. multivorans WS-FJ9 strain did not possess the BCESM and cblA virulence genes. Scanning electron microscopy revealed that the colonization of the WS-FJ9 strain reached 1.4 × 10 colony forming units (cfu) g rhizosphere soil on day 77 post-inoculation. The B. multivorans WS-FJ9 strain could colonize the rhizosphere as well as the root tissues and cells of poplars. Greenhouse evaluations in both sterilized and non-sterilized soils indicated that B. multivorans WS-FJ9 significantly promoted growth in height, root collar diameter, and plant biomass of inoculated poplar seedlings compared with controls. Phosphorus contents of roots and stems of treated seedlings were 0.57 and 0.55 mg g higher than those of the controls, respectively. Phosphorus content was lower in the rhizosphere soils by an average of 1.03 mg g compared with controls. The results demonstrated that B. multivorans WS-FJ9 is a nonpathogenic strain that could colonize the roots and significantly promote the growth of poplar seedlings. [ABSTRACT FROM AUTHOR]

Burkholderia cepacia complex (Bcc) is a group of bacteria with conflicting biological characteristics, which make them simultaneously beneficial and harmful to humans. They have been exploited for biocontrol, bioremediation, and plant growth promotion. However, their capacity as opportunistic bacteria that infect humans restricts their biotechnological applications. Therefore, the risks of using these bacteria should be assessed. In this study, Burkholderia multivorans WS-FJ9 originally isolated from pine rhizosphere, which was shown to be efficient in solubilizing phosphate, was evaluated with respect to its biosafety, colonization in poplar rhizosphere, and growth-promoting effects on poplar seedlings. Pathogenicity of B. multivorans WS-FJ9 on plants was determined experimentally using onion and tobacco as model plants. Onion bulb inoculated with B. multivorans WS-FJ9 showed slight hypersensitive responses around the inoculation points, but effects were not detectable based on the inner color and odor of the onion. Tobacco leaves inoculated with B. multivorans WS-FJ9 exhibited slightly water-soaked spots around the inoculation points, which did not expand or develop into lesions even with repeated incubation. Pathogenicity of the strain in alfalfa, which has been suggested as an alternative Bcc model for mice, was not detectable. Results from gene-specific polymerase chain reactions showed that the tested B. multivorans WS-FJ9 strain did not possess the BCESM and cblA virulence genes. Scanning electron microscopy revealed that the colonization of the WS-FJ9 strain reached 1.4 × 10 4 colony forming units (cfu) g −1 rhizosphere soil on day 77 post-inoculation. The B. multivorans WS-FJ9 strain could colonize the rhizosphere as well as the root tissues and cells of poplars. Greenhouse evaluations in both sterilized and non-sterilized soils indicated that B. multivorans WS-FJ9 significantly promoted growth in height, root collar diameter, and plant biomass of inoculated poplar seedlings compared with controls. Phosphorus contents of roots and stems of treated seedlings were 0.57 and 0.55 mg g −1 higher than those of the controls, respectively. Phosphorus content was lower in the rhizosphere soils by an average of 1.03 mg g −1 compared with controls. The results demonstrated that B. multivorans WS-FJ9 is a nonpathogenic strain that could colonize the roots and significantly promote the growth of poplar seedlings. [ABSTRACT FROM AUTHOR]

A number of soil microorganisms can convert insoluble forms of phosphorus (P) to an accessible form to increase plant yields. Phytate is such a large kind of insoluble organic phosphorus that plants cannot absorb directly in soil, so the objectives of this study were to isolate, screen phytate-degrading rhizobacteria (PDRB), and to select potential microbial inocula that could increase the P uptake by plants. In this study, a total of 24 soil samples were collected from natural habitats of eight poplar and pine planting areas from the eastern to southern China. 17 PDRB strains were preliminarily screened from the rhizosphere soil of poplars and pines by the visible decolorization in the phytate selective medium. The highest ratio of the total diameter (colony + halo zone) to the colony diameter of the isolates was JZ-GX1, 3.85. Afterward, 17 PDRB strains were further determined for their abilities to degrade sodium phytate based on the amount of liberated inorganic P in liquid phytate specific medium. The results showed that the phytase ability of the three highest PDRB strains: JZ-GX1, JZ-DZ1 and JZ-ZJ1 were up to 2.58, 2.36 and 2.24 U/mL, respectively, much better than most of the bacteria reported in previous studies. In the soil-plant experiment, compared to CK, the best three strains of PDRB all could significantly promote growth of poplar and Masson pine under container growing. The three efficient PDRB strains were identified as follow: JZ-GX1, Rahnella aquatilis, both JZ-DZ1 and JZ-ZJ1 being autofluorescent, Pseudomonas fluorescens, by 16S rDNA gene sequencing technology, Biolog Identification System and biological characterization. The present study suggests that the three screened PDRB strains would have great potential application as biological fertilizers in the future. [ABSTRACT FROM AUTHOR]

The content of NO and HO as well as the activities of nitric oxide synthase (NOS)-like and nitrate reductase (NR) were monitored in the needles of Pinus thunbergii infected by Bursaphelenchus xylophilus. The results showed that the content of NO increased significantly only 8 h after the invasion of B. xylophilus, while HO increased 12 h after invasion. NO donor SNP could promote and NO scavenger cPTIO could prevent the production of NO and HO. The content of NO changed earlier than that of HO. In addition, the symptoms appeared 9, 5 and 12 days, respectively, after the inoculation with B. xylophilus, SNP pre-treatment and cPTIO pre-treatment followed by B. xylophilus infection. After B. xylophilus infection, the content of NO in P. thunbergii changed fiercely more earlier than the appearance of external symptoms, which indicated that the content of NO was related with the appearance and the development of the symptoms. The treatment with l-NNA (NOS inhibitor) inhibited the content of NO significantly, whereas, NaWO (NR inhibitor) had no effect. The further analysis of NOS revealed that NO changed in consistent with cNOS activity. To sum up, NO, as the upstream signal molecule of HO, was involved in the pine early response to the invasion of B. xylophilus and influenced the accumulation of the content of HO. Moreover, NOS-like rather than NR was responsible for the endogenous NO generation, which was modulated by cNOS during the interaction between P. thunbergii and B. xylophilus. Key message NO is involved in early response of P. thunbergii to the invasion of B. xylophilus and NOS is the key enzyme responsible for NO generation in P. thunbergii. [ABSTRACT FROM AUTHOR]

The present greenhouse study was undertaken to evaluate the effects of co-inoculating the ectomycorrhizal (ECM) fungus Boletus edulis with the mycorrhiza helper bacterium Bacillus cereus HB12 or HB59 on the growth and nutrient uptake of Pinus thunbergii. The inoculation with mycorrhiza helper bacterium significantly ( P ≤ 0.05) increased the ectomycorrhizal colonization. Treatments with dual inoculum (the mycorrhiza helper bacterium plus mycorrhiza) significantly ( P ≤ 0.05) increased the P. thunbergii growth. Bacteria-mycorrhizae interactions resulted in a great utilization of phosphate and potassium. The single inoculation resulted in a higher root activity than the control while the co-inoculation led to the highest root activity. The 6-CFDA staining assay showed that B. cereus enhanced fungal activity in ectomycorrhizal symbiosis. The results conclusively suggest that B. cereus isolated from the rhizosphere of P. thunbergii can potentially be used as individual inoculant or co-inoculated with ECM fungi to increase the production in sustainable ecological systems. These results support the potential use of B. cereus (HB12 or HB59) and B. edulis as mixed inoculants stimulating growth of P. thunbergii. [ABSTRACT FROM AUTHOR]

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a disastrous pathogen of the pine forests in East Asia and Europe. Plant quarantine is one of the most important ways to prevent its infection in current situation. A nested polymerase chain reaction (PCR) assay targeting the topoisomerse I gene has been developed to detect PWN in this study. To assess the specificity of the assay, 44 morphologically characterized nematode isolates including B. xylophilus, B. mucronatus, B. hofmanni, Seinura wuae, S. lii and Aphelenchoides macronucleatus were tested. Positive reactions characterized by amplification product of 509 bp were shown from all isolates of PWN. The nested PCR assay can detect 50 femtogram (fg) of template DNA or one individual nematode, as small as an egg. The validity was evaluated by analyzing the nematode samples extracted from the nematode-infested wood in the field. These results show that the assay is a specific, sensitive method for detection of PWN with the potential in relation to the pest risk assessment and quarantine regulations. [ABSTRACT FROM AUTHOR]