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Verticillium dahliae is among the most devastating fungal pathogens, causing significant economic harm to agriculture and forestry. To address this problem, researchers have focused on eliciting systemic resistance in host plants through utilizing volatile organic compounds (VOCs) produced by biological control agents. Herein, we meticulously measured the quantity of V. dahliae pathogens in plants via RTqPCR, as well as the levels of defensive enzymes and pathogenesis-related (PR) proteins within plants. Finally, the efficacy of VOCs in controlling Verticillium wilt in cotton was evaluated. Following treatment with Pseudomonas aurantiaca ST-TJ4, the expression of specific VdEF1-α genes in cotton decreased significantly. The incidence and disease indices also decreased following VOC treatment. In cotton, the salicylic acid (SA) signal was strongly activated 24 h posttreatment; then, hydrogen peroxide (H 2 O 2 ) levels increased at 48 h, and peroxidase (POD) and catalase (CAT) activities increased to varying degrees at different time points. The malondialdehyde (MDA) content and electrolyte leakage in cotton treated with VOCs were lower than those in the control group, and the expression levels of chitinase (CHI) and PR genes (PR10 and PR17), increased at various time points under the ST-TJ4 treatment. The activity of phenylalanine ammonia lyase (PAL) enzymes in cotton treated with VOCs was approximately 1.26 times greater than that in control plants at 24 h，while the contents of phenols and flavonoids increased significantly in the later stage. Additionally, 2-undecanone and 1-nonanol can induce a response in plants that enhances disease resistance. Collectively, these findings strongly suggest that VOCs from ST-TJ4 act as elicitors of plant defence and are valuable natural products for controlling Verticillium wilt., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests, (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Purpose: Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a type of pediatric HLH that occurs frequently in Asia. Although immunochemotherapy based on etoposide and hormone has improved survival rates, there are still about 30% of HLH patients that do not respond. The objective of the article is to examine the efficacy and safety of programmed cell death protein 1 (PD-1) inhibitors for children with relapsed/refractory (r/r) EBV-HLH., Methods: A retrospective case note review of four pediatric patients with r/r EBV-HLH who were treated with PD-1 inhibitors at Sun Yat-sen Memorial Hospital, Sun Yat-sen University., Results: All four patients responded to PD-1 inhibitors and achieved partial response after their first infusion. Plasma EBV DNA copy number and HLH-related monitoring indicators decreased in all of these patients. All patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT), and two were still alive at the last follow-up on December 30, 2022. Two patients died because of transplantation-related complications. Serious side effects included increased liver enzymes and edema in two patients., Conclusion: PD-1 inhibitors are an effective salvage therapy and can provide a bridge to allo-HSCT for pediatric patients with r/r EBV-HLH. However, side effects should be monitered., (© 2024 Wiley Periodicals LLC.)

Pinus is an important economic tree species, but pine wilt disease (PWD) seriously threatens the survival of pine trees. PWD caused by Bursaphelenchus xylophilus is a major quarantine disease worldwide that causes significant economic losses. However, more information about its molecular pathogenesis is needed, resulting in a lack of effective prevention and treatment measures. In recent years, effectors have become a hot topic in exploring the molecular pathogenic mechanism of pathogens. Here, we identified a specific effector, BxNMP1, from B. xylophilus . In situ hybridization experiments revealed that BxNMP1 was specifically expressed in dorsal gland cells and intestinal cells, and RT-qPCR experiments revealed that BxNMP1 was upregulated in the early stage of infection. The sequence of BxNMP1 was different in the avirulent strain, and when BxNMP1 -silenced B. xylophilus was inoculated into P. thunbergii seedlings, the disease severity significantly decreased. We demonstrated that BxNMP1 interacted with the thaumatin-like protein PtTLP-L2 in P. thunbergii . Additionally, we found that the β-1,3-glucanase PtGLU interacted with PtTLP-L2. Therefore, we hypothesized that BxNMP1 might indirectly interact with PtGLU through PtTLP-L2 as an intermediate mediator. Both targets can respond to infection, and PtTLP-L2 can enhance the resistance of pine trees. Moreover, we detected increased salicylic acid contents in P. thunbergii seedlings inoculated with B. xylophilus when BxNMP1 was silenced or when the PtTLP-L2 recombinant protein was added. In summary, we identified a key virulence effector of PWNs, BxNMP1. It positively regulates the pathogenicity of B. xylophilus and interacts directly with PtTLP-L2 and indirectly with PtGLU. It also inhibits the expression of two targets and the host salicylic acid pathway. This study provides theoretical guidance and a practical basis for controlling PWD and breeding for disease resistance.

Bursaphelenchus xylophilus is the pathogen of pine wilt disease, which can devastate the pine forest ecosystem. Usually, plant cells generate reactive oxygen species (ROS) as a defensive substance or signalling molecules to resist the infection of nematodes. However, little is known about how B. xylophilus effectors mediate the plant ROS metabolism. Here, we identified a pioneer B. xylophilus Prx3-interacting effector 1 (BxPIE1) expressed in the dorsal gland cells and the intestine. Silencing of the BxPIE1 gene resulted in reduced nematode reproduction and a delay in disease progression during parasitic stages, with the upregulation of pathogenesis-related (PR) genes PtPR-3 (class Ⅳ chitinase) and PtPR-9 (peroxidase). The protein-protein interaction assays further demonstrated that BxPIE1 interacts with a Pinus thunbergii class III peroxidase (PtPrx3), which produces H 2 O 2 under biotic stress. The expression of BxPIE1 and PtPrx3 was upregulated during the infection stage. Furthermore, BxPIE1 effectively inhibited H 2 O 2 generating from class III peroxidase and ascorbate can recover the virulence of siBxPIE1-treated B. xylophilus by scavenging H 2 O 2 . Taken together, BxPIE1 is an important virulence factor, revealing a novel mechanism utilized by nematodes to suppress plant immunity., (© 2024 John Wiley & Sons Ltd.)

Background: Palliative care is fraught with numerous challenges when it comes to conducting practical teaching as it involves caring for people facing the complexities of end-of-life and death. Insufficient clinical practice hinders nursing students from mastering knowledge, attitude and ability of hospice care. Virtual clinical simulation has demonstrated its effectiveness as a valuable educational tool in nursing. However, there is a dearth of evidence supporting its utilization in the context of palliative care practice education., Objective: To develop a virtual clinical simulation education system and assess its impact on enhancing nursing students' knowledge, ability, and attitudes toward palliative care., Design: A single-group pretest-posttest design and focus group interviews were employed., Setting: The study was conducted at a medical university in southwest China., Participants: A total of 76 third-year nursing students participated., Methods: Participants underwent a 1-hour learning session using the virtual clinical simulation education system. Pre-test and post-test evaluations were conducted to assess the participants' knowledge, ability, and attitudes toward palliative care. Survey questionnaire was administered to gauge the students' acceptance and perception of virtual clinical simulation. Focus group interviews were integrated to gain insight into students' subjective perceptions and feedback on the virtual clinical simulation., Results: There were notable enhancements in the students' overall scores of palliative care knowledge, ability, and attitudes after the learning session. Students positively evaluated the usefulness and usability of virtual clinical simulation. Students' feedback regarding virtual clinical simulation can be categorized into four themes: the value of virtual clinical simulation education system, its role as a complement to clinical practice teaching, the enjoyment and accessibility of learning, and the technological challenges encountered., Conclusion: Virtual clinical simulation is an effective learning tool in palliative care practice education, which has the potential to enhance students' knowledge, ability, and attitudes toward palliative care., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Lipase is involved in lipid hydrolysis, which is related to nematodes' energy reserves and stress resistance. However, the role of lipases in Bursaphelenchus xylophilus, a notorious plant-parasitic nematode responsible for severe damage to pine forest ecosystems, remains largely obscure. Here, we characterized a class III lipase as a candidate effector and named it BxLip-3. It was transcriptionally up-regulated in the parasitic stages of B. xylophilus and specifically expressed in the oesophageal gland cells and the intestine. In addition, BxLip-3 suppressed cell death triggered by the pathogen-associated molecular patterns PsXEG1 and BxCDP1 in Nicotiana benthamiana, and its Lipase-3 domain is essential for immunosuppression. Silencing of the BxLip-3 gene resulted in a delay in disease onset and increased the activity of antioxidant enzymes and the expression of pathogenesis-related (PR) genes. Plant chitinases are thought to be PR proteins involved in the defence system against pathogen attack. Using yeast two-hybrid and co-immunoprecipitation assays, we identified two class I chitinases in Pinus thunbergii, PtChia1-3 and PtChia1-4, as targets of BxLip-3. The expression of these two chitinases was up-regulated during B. xylophilus inoculation and inhibited by BxLip-3. Overall, this study illustrated that BxLip-3 is a crucial virulence factor that plays a critical role in the interaction between B. xylophilus and host pine., (© 2023 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Persistence in the soil is a function of seed physiology, particularly non-germination and inherent lifespan. However, for seeds with mechanical dormancy, non-germination is also a function of the composition and activity of the soil microbiota. We attempted to screen out microorganisms in the soil that can specifically and rapidly decompose the hard fruit pericarps of Tilia miqueliana Maxim., a unique native tree species in China. Using the classical replica plating method, more than 100 different culturable microorganisms that could rapidly erode the pericarp were collected from the surface of pericarps under different culture conditions. At the same time, we successfully extended the concept of metagenomics and applied it to the identification of mixed artificial cultures. The decomposition process of the pericarps in soil was also simulated artificially. The physical and chemical data suggested a potential mechanism of microbial scarification and cracking in pericarp, whilst the embryos inside the eroded fruits retained good viability. Our discoveries could pave the way for the removal of physical and mechanical obstacles that prevent hard coat seeds from germinating. We anticipate that the use of this technology will improve the germination of other hard coat seeds. More research is needed to investigate the impacts on other seeds. The findings of this research can inform the design of experiments on the seed ecology of persistence., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wu, Sun, Pritchard, Shen, Wu and Peng.)

Pine wilt disease (caused by the nematode Bursaphelenchus xylophilus) is extremely harmful to pine forests in East Asia. As a low-resistance pine species, Pinus thunbergii is more vulnerable to pine wood nematode (PWN) than Pinus densiflora and Pinus massoniana. Field inoculation experiments were conducted on PWN-resistant and -susceptible P. thunbergii, and the difference in transcription profiles 24 h after inoculation was analyzed. We identified 2603 differentially expressed genes (DEGs) in PWN-susceptible P. thunbergii, while 2559 DEGs were identified in PWN-resistant P. thunbergii. Before inoculation, DEGs between PWN-resistant and PWN-susceptible P. thunbergii were enriched in the REDOX (Oxidation-Reduction) activity pathway (152 DEGs), followed by the oxidoreductase activity pathway (106 DEGs). After inoculation with PWN, however, the opposite was observed; DEGs were enriched in the oxidoreductase activity pathway (119 DEGs), followed by the REDOX activity pathway (84 DEGs). Before inoculation, according to the metabolic pathway analysis results, we found more genes upregulated in phenylpropanoid metabolic pathways and enriched in lignin synthesis pathways; cinnamoyl-CoA reductase-coding genes related to lignin synthesis were upregulated in PWN-resistant P. thunbergii and downregulated in PWN-susceptible P. thunbergii, and the lignin content was always higher in resistant than in susceptible P. thunbergii. These results reveal distinctive strategies of resistant and susceptible P. thunbergii in dealing with PWN infections., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Utilization of rhizosphere microorganisms to improve plant growth and salt tolerance has recently attracted widespread attention. The growth and salt tolerance of willows inoculated with Bacillus cereus JYZ-SD2 and Peniophora cinerea XC were studied under different salt stress conditions. The results showed that the chlorophyll content of willow cuttings inoculated with the XC strain increased significantly by 51.27%. After salt stress of willow cuttings inoculated with B. cereus JYZ-SD2 and P. cinerea XC (solely or in combination), the amount of sodium in the roots from the epidermis to the pericycle decreased and the content of sodium in the pericycle was significantly lower than that of the uninoculated willow, while the proportion of potassium increased. Willow cuttings inoculated with microorganisms showed increased activity of SOD and POD. At the salt concentration of 100 mmol/L, the highest SOD activity was found in B. cereus JYZ-SD2-inoculated willows, with 59.88% increase compared to uninoculated willows; the highest POD activity was found in P. cinerea XC and B. cereus JYZ-SD2 co-inoculated willows, with 51.05% increase compared to uninoculated willows. The Na-K-ATPase and Ca-Mg-ATPase activities of inoculated P. cinerea XC willow cuttings were also 59.38% and 60% higher than that of uninoculated willows, respectively. The qPCR analysis showed that the expression of vp2 gene in the microorganism-inoculated willow leaves was always higher than that in willow alone. The expression of vp2 gene in P. cinerea XC-inoculated willow cuttings was 270.81% higher than that in uninoculated willows. Further observation of the ultrastructure of root cells under salt stress revealed that most of the vesicles in the root tip cells of willow were intact and secreted phagocytic vesicles to absorb sodium ions in the cytoplasm. This study shows that the combined beneficial fungi and rhizosphere-promoting bacteria inoculation technology as a practical biotechnological approach to enhance the growth of willows in salt-affected soils., Competing Interests: Conflicts of Interest The authors declare no conflicts of interest., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Pine wilt disease, caused by Bursaphelenchus xylophilus , results in tremendous economic loss in conifer production every year. To disturb the host immune responses, plant pathogens secrete a mass of effector proteins that facilitate the infection process. Although several effectors of B. xylophilus have been identified, detailed mechanisms of their functions remain largely unexplored. Here, we reveal two novel B. xylophilus Kunitz effectors, named BxKU1 and BxKU2, using different infection strategies to suppress immunity in Pinus thunbergii . We found that both BxKU1 and BxKU2 could suppress PsXEG1-triggered cell death and were present in the nucleus and cytoplasm in Nicotiana benthamiana . However, they had different three-dimensional structures and various expression patterns in B. xylophilus infection. In situ hybridization experiments showed that BxKU2 was expressed in the esophageal glands and ovaries, whereas BxKU1 was only expressed in the esophageal glands of females. We further confirmed that the morbidity was significantly decreased in P. thunbergii infected with B. xylophilus when BxKU1 and BxKU2 were silenced. The silenced BxKU2I, but not BxKU1 , affected the reproduction and feeding rate of B. xylophilus . Moreover, BxKU1 and BxKU2 targeted to different proteins in P. thunbergii , but they all interacted with thaumatin-like protein 4 (TLP4) according to yeast two-hybrid screening. Collectively, our study showed that B. xylophilus could incorporate two Kunitz effectors in a multilayer strategy to counter immune response in P. thunbergii , which could help us better understand the interaction between plant and B. xylophilus .

Background: In general, medical staff who work in nuclear medicine should be entirely safe in their professional environment. Nevertheless, we already know that the working environment of the nuclear medicine staff is not completely safe due to the handling of high amounts of radionuclides for diagnostic and therapy applications, which is especially relevant for 131 I (as a non-sealed source)., Purpose: The goal of this study was to assess the inhaled 131 I thyroid dose in nuclear medicine workers and to introduce a simple method for internal exposure monitoring., Methods: Using 2-IN * 2-in NaI (Tl) scintillation spectrometer and its supporting software (InSpector Maintenance Utility and Genie 2000), from 2019 to 2021, internal thyroid irradiation monitoring, an internal thyroid irradiation monitoring investi A NaI (Tl) scintillation spectrometer and its sugation was carried out for 3 consecutive years, between 2019 and 2021, in staff members of nuclear medicine departments engaged with iodine therapy., Results: 131 I activity was found highest in the thyroid of nuclear medicine workers involved with the manual packaging and delivery of the radioisotope, while it was not detected in staff members involved with the automatic packaging and drug delivery. The activity range was found to be 30.00 ± 6.60-6070.00 ± 1335.40 Bq for the exposed personnel, and the estimated dose was 0.05-6.77 mSv. In 2021, three workers had an annual equivalent dose above 5 mSv., Conclusion: By monitoring the thyroid 131 I in staff members of the nuclear medicine department, it was found that there are 131 I internal occupational exposure risks. The best solution is automatic packaging and drug delivery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Li, Zhang, Zhang, Liu, Wu, Niu and Zhang.)

The pinewood nematode, Bursaphelenchus xylophilus , has been determined as one of the world's top ten plant-parasitic nematodes. It causes pine wilt, a progressive disease that affects the economy and ecologically sustainable development in East Asia. B. xylophilus secretes pathogenic proteins into host plant tissues to promote infection. However, little is known about the interaction between B. xylophilus and pines. Previous studies reported transthyretin proteins in some species and their strong correlation with immune evasion, which has also been poorly studied in B. xylophilus . In this study, we cloned and functionally validated the B. xylophilus pathogenic protein BxTTR-52, containing a transthyretin domain. An in situ hybridization assay demonstrated that BxTTR-52 was expressed mainly in the esophageal glands of B. xylophilus . Confocal microscopy revealed that BxTTR-52-RFP localized to the nucleus, cytoplasm, and plasma membrane. BxTTR-52 recombinant proteins produced by Escherichia coli could be suppressed by hydrogen peroxide and antioxidant enzymes in pines. Moreover, silencing BxTTR-52 significantly attenuated the morbidity of Pinus thunbergii infected with B. xylophilus . It also suppressed the expression of pathogenesis-related genes in P. thunbergii . These results suggest that BxTTR-52 suppresses the plant immune response in the host pines and might contribute to the pathogenicity of B. xylophilus in the early infection stages.

A 2D Co-MOF, {[Co 2 (L 2- ) 2 (bipy)](DMA)·2H 2 O} n (Co-1, H 2 L = 2,5-thienedioic acid; bipy = 2,2'-bipyridine; DMA = N , N '-dimethyl acetamide), was synthesized by hydrothermal method. Co-1 has excellent air stability. When modifying the surface of a glassy carbon electrode (GCE) with Co-1, the obtained electrochemical senor Co-1/GCE shows excellent sensitivity towards 1,3-dinitrobenzene ( m -DNB) and 2,4-dinitroaniline (2,4-DNA), although the electrochemical conductivity of Co-1 is not that good. The detection limits were as low as 0.0286 μM and 0.161 μM, respectively. DFT studies showed that the main interaction between Co-1 and the guest molecules is via hydrogen bonding, formed by the -NO 2 group and the coordinated H 2 O molecule from the Co-1 skeleton. Furthermore, the characteristic signals of both m -DNB and 2,3-DNA can still be observed in a mimicked industrial waste-water system containing 17 kinds of organic interferents, indicating high selectivity of the Co-1/GCE sensor.

Pseudocydonia sinensis is a Chinese ornamental plant with great landscaping value. Its fruit is additionally used for medicinal purposes (Lim 2012). In June 2020, a leaf spot disease was observed in the campus of Nanjing Forestry University (32°04'34.53″N 118°48'42.06″E). The symptoms began with irregular red-brown spots, which gradually enlarged, extended and overlapped, with an incidence of 85% (29/34 trees). Pieces of leaf tissue (3 to 4 mm²) from the lesion margins were surface-sterilized with 75% ethanol for 30 s and 1% NaClO for 90 s. Subsequently, the tissues were rinsed with sterile H2O, placed on potato dextrose agar (PDA) medium and incubated at 25℃ for 5 days. The same fungus was isolated from 90% of tissues. Pure cultures were obtained by monosporic isolation.The representative isolate NJMG 5-7 was used for morphological and molecular characterization. The growing fungal colony on PDA was initially white, but gradually turned grey. Pycnidia formation was observed on PDA supplemented with alfalfa stems. The pycnidia produced two different types of conidia, α and β, which ooze out in yellow creamy mucilaginous masses. Conidiophores were hyaline, cylindrical and smooth, 16.8 to 33.1 × 1.5 to 2.6 μm (n=30). Conidiogenous cells were 13.6 to 29.3 × 1.5 to 2.7 μm (n=30). The α-conidia were, unicellular, hyaline elliptical or fusiform, bi-guttulate, 6.5 to 9.2 × 1.8 to 3.3 µm (n = 50). The β-conidia were hyaline, aseptate, without guttules, filiform, curved, with obtuse ends, 12.5 to 25 × 1.0 to 1.8 µm (n = 50). To verify species identity, the partial sequences of the internal transcribed spacer (ITS) region, and calmodulin (CAL), translation elongation factor 1 alpha (EF1-a), and beta-tubulin genes (TUB) were amplified from isolate NJMG 5-7 with primers ITS1/ITS4 (White et al. 1990), CAL-228F/CAL-737R (Carbone & Kohn 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The sequences were deposited in GenBank (OP223050 for ITS, OP252809 for CAL, OP252807 for EF1-a, and OP252808 for TUB). A BLAST search of GenBank showed that ITS, CAL, EF1-a and TUB sequences of NJMG 5-7 were similar to those of D. eres CBS 138594 (99% identity), AR5193 (99%), AR5193 (99%) and MG281193 (100%), respectively. The morphological and molecular results identified the isolate as D. eres (Feng et al. 2015). To fulfill Koch's postulates, a pathogenicity test was conducted using three P. sinensis plants. Six leaves from each tree were wounded and inoculated with mycelial plugs (about 4 mm in diameter) of D. eres from a 3-day-old culture grown on PDA. Inoculations with sterile PDA plugs on different leaves of the same tree were used as controls. All inoculated leaves were enclosed in plastic bags together with a wet cotton ball inside. Sterile H2O was sprayed into the plastic bags to keep moisture conditions. Five days later, all inoculated points showed lesions similar to those previously observed in the field, whereas controls were asymptomatic. The pathogen was successfully reisolated from the inoculated symptomatic parts on PDA and identified from its morphology, thus fulfilling Koch's postulates. This fungus can cause a variety of diseases. To our knowledge, this is the first report of D. eres causing leaf spots on P. sinensis in the world. These findings provide a foundation for future studies on the epidemiology and control of this newly emerging disease.

Background: In the present study, we analyzed radiation injuries to Chinese workers exposed to low-dose radiation. We discuss the relationships between dose and injury., Methods: This study randomly selected 976 radiation workers who underwent occupational health monitoring. The radiation workers were divided into 5 different types of work: radiation diagnosis, radiation therapy, interventional therapy, nuclear medicine, and industrial inspection. This research was approved by the Bioethics Committee at the Gansu Provincial Center for Disease Control and Prevention., Results: The average annual cumulative dose to interventional radiation workers was the highest, i.e., 0.86 mSv. The detection rate of lens opacity was 37%, but 99.70% of lens opacities occurred in the peripheral cortex. Posterior subcapsular opacification was detected less than 1.00% of the time. The rate of chromosomal aberrations was highest for radiological workers with more than 20 years of service. Annual cumulative dose reached 2.04 mSv, and the monitoring dose for 3 months was as high as 1.62 mSv. Dicentric chromosomes were also detected. The manual packaging and drug delivery nuclear medicine staffs totaled 14 individuals. 131 131 was detected in the thyroids of 4 workers (28.57%). The detection rate of thyroid 131 I was higher in the hand-packed and administered group than in the automatic administration group., Conclusion: Radiation workers exposed to low doses of radiation can sustain injuries. Interventional radiology workers receive the highest doses and sustain the most significant effects. This study suggests that chromosome aberration analysis is an important index in occupational health monitoring of radiological workers. Monitoring of internal radiation exposure cannot be ignored for nuclear medicine staff., Competing Interests: The authors declare no conflicts of interest. Gang Liu and Rong Zhang are co-first authors with equal contributions., (Copyright © 2022 Health Physics Society.)

Volatile organic compounds (VOCs) produced by microorganisms are considered promising environmental-safety fumigants for controlling soil-borne diseases. Verticillium dahliae , a notorious fungal pathogen, causes economically important wilt diseases in agriculture and forestry industries. Here, we determined the antifungal activity of VOCs produced by Trichoderma koningiopsis T2. The VOCs from T. koningiopsis T2 were trapped by solid-phase microextraction (SPME) and tentatively identified through gas chromatography-mass spectrometry (GC/MS). The microsclerotia formation, cell wall-degrading enzymes and melanin synthesis of V. dahliae exposed to the VOC mixtures and selected single standards were examined. The results showed that the VOCs produced by strain T2 significantly inhibited the growth of V. dahliae mycelium and reduced the severity of Verticillium wilt in tobacco and cotton. Six individual compounds were identified in the volatilome of T. koningiopsis T2, and the dominant compounds were 3-octanone, 3-methyl-1-butanol, butanoic acid ethyl ester and 2-hexyl-furan. The VOCs of strain T2 exert a significant inhibitory effect on microsclerotia formation and decreased the activities of pectin lyase and endo-β-1,4-glucanase in V. dahliae . VOCs also downregulated the VdT3HR , VdT4HR , and VdSCD genes related to melanin synthesis by 29. 41-, 10. 49-, and 3.11-fold, respectively. Therefore, T. koningiopsis T2 has potential as a promising biofumigant for the biocontrol of Verticillium wilt disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kong, Ni, Wang and Wu.)

Plant growth-promoting rhizobacteria are important for improving plant iron nutrition, but the interactions among inoculants, host plants and soil microorganisms have not been greatly explored. Rahnella aquatilis JZ-GX1 was applied to treat the increasingly serious iron deficiency chlorosis in Cinnamomum camphora , and the resulting improvement in chlorosis was determined by assessing the contents of chlorophyll, active iron, Fe 2+ and antioxidant enzymes in leaves, the effects on the soil microbial community and the metabolism in the rhizosphere by high-throughput sequencing techniques and liquid chromatography-mass spectrometry (LC-MS). The results showed that inoculation with JZ-GX1 significantly increased the chlorophyll content of C. camphora , which promoted the redistribution of active iron in roots and leaves, increased the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX), and thus reduced membrane damage in iron-deficient C. camphora caused by reactive oxygen species. According to genome prediction and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis, the JZ-GX1 strain could secrete desferrioxamine (DFO), and the concentration of DFO in C. camphora rhizosphere was 21-fold higher than that in uninoculated soil. The exogenous application of DFO increased the SPAD and Fe 2+ contents in leaves. In addition, the inoculant affected the fungal community structure and composition in the C. camphora rhizosphere soil and increased the abundances of specific taxa, such as Glomus , Mortierella , Trichoderma, and Penicillium . Therefore, R. aquatilis JZ-GX1 application promoted iron absorption in C. camphora trees by secreting DFO and alleviated iron deficiency chlorosis through interactions with the local fungal community., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kong, Wang, Lu, Li, Zhang and Wu.)

Phytophthora cinnamomi is an important plant pathogen that is widely distributed worldwide and has caused serious ecological damage and significant economic losses in forests and plantations in many countries. The use of plant growth-promoting rhizobacteria is an effective and environmentally friendly strategy for controlling diseases caused by P. cinnamomi . In this study, we investigated the antagonistic mechanism of Pseudomonas aurantiaca ST-TJ4 against P. cinnamomi through different antagonistic approaches, observations of mycelial morphology, study of mycelial metabolism, and identification of antagonistic substances. The results showed that Pseudomonas aurantiaca ST-TJ4 was able to significantly inhibit mycelial growth, causing mycelial deformation and disrupting internal cell structures. Additionally, pathogen cell membranes were damaged by ST-TJ4, and mycelial cell content synthesis was disrupted. Ultraperformance liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry analyses showed that phenazine compounds and 2-undecanone were the main antagonistic components. The ammonia produced by the ST-TJ4 strain also contributed to the inhibition of the growth of P. cinnamomi . In conclusion, our results confirm that Pseudomonas aurantiaca ST-TJ4 can inhibit P. cinnamomi through multiple mechanisms and can be used as a biological control agent for various plant diseases caused by P. cinnamomi .

Iron deficiency causes chlorosis and growth inhibition in Cinnamomum camphora , an important landscaping tree species. Siderophores produced by plant growth-promoting rhizobacteria have been widely reported to play an indispensable role in plant iron nutrition. However, little to date has been determined about how microbial siderophores promote plant iron absorption. In this study, multidisciplinary approaches, including physiological, biochemical and transcriptome methods, were used to investigate the role of deferoxamine (DFO) in regulating Fe availability in C. camphora seedlings. Our results showed that DFO supplementation significantly increased the Fe 2+ content, SPAD value and ferric-chelate reductase (FCR) activity in plants, suggesting its beneficial effect under Fe deficiency. This DFO-driven amelioration of Fe deficiency was further supported by the improvement of photosynthesis. Intriguingly, DFO treatment activated the metabolic pathway of glutathione (GSH) synthesis, and exogenous spraying reduced glutathione and also alleviated chlorosis in C. camphora . In addition, the expression of some Fe acquisition and transport-related genes, including CcbHLH , CcFRO6 , CcIRT2, CcNramp5 , CcOPT3 and CcVIT4 , was significantly upregulated by DFO treatment. Collectively, our data demonstrated an effective, economical and feasible organic iron-complexing agent for iron-deficient camphor trees and provided new insights into the mechanism by which siderophores promote iron absorption in plants.

The migratory plant-parasitic nematode Bursaphelenchus xylophilus is the pathogen of the pine wilt disease (PWD), causing serious damage to pine forests in China. During the process of plant resistance to multiple pathogens, plant immunity plays a key role. In this current study, the pathogen-associated molecular pattern (PAMP) BxCDP1 in B. xylophilus has been identified, but the host target protein of BxCDP1 and its key amino acid region inducing the plant immunity have yet to be elucidated. We found that BxCDP1 could trigger superoxide production, H 2 O 2 production, and callose deposits. A RING-H2 finger protein 1 (RHF1) of Pinus thunbergii was screened and characterized as a target protein of BxCDP1 by yeast two-hybrid and co-immunoprecipitation (Co-IP). Moreover, two peptides (namely M9 and M16) proved to be key regions of BxCDP1 to induce PAMP-triggered immunity (PTI) in Nicotiana benthamiana , which also induced the expression of pathogenesis-related (PR) genes ( PtPR-3 , PtPR-4 , and PtPR-5 ) in P. thunbergii and enhanced the resistance of the host to B. xylophilus . These results indicate that BxCDP1 plays a critical role in the interaction between B. xylophilus and P. thunbergii , and both peptides M9 and M16 have the potential to be developed and utilized as immune inducers of pines against B. xylophilus in future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hu, Wu, Wen, Ye, Qiu, Rui and Zhang.)

Liriodendron chinense × tulipifera black spot is a newly discovered disease that causes yellowing and early shedding of leaves, affecting the growth of Liriodendron trees, and significantly reducing their ornamental value as a garden species. The pathogen responsible for this disease, and how it can be prevented and controlled, are not clear. In this study, the occurrence of this disease was first investigated according to Koch's postulates, and the primary pathogens causing Liriodendron black spot were determined to be Colletotrichum gloeosporioides and Alternaria alternata . Biocontrol strains antagonistic to these two pathogens were then screened from the leaf microorganisms of L . chinense × tulipifera , and a preliminary investigation of the biological control of Liriodendron black spot was performed. Through the screening of antagonistic microorganisms on the leaf surface of L . chinense × tulipifera , the strain Trichoderma koningiopsis T2, which displayed strong antagonism against C . gloeosporioides and A . alternata , was obtained. The T2 strain could inhibit the growth of the two pathogens via three mechanisms: hyperparasitism, volatile and nonvolatile metabolite production, and environmental acidification. The biocontrol experiments in the greenhouse and field showed that initial spraying with a T . koningiopsis T2 spore suspension followed by the two pathogens resulted in the lowest disease incidence. These results confirmed the black spot pathogens of L . chinense × tulipifera , clarified the antagonistic mechanism of T . koningiopsis T2 against the two pathogens, and provided a theoretical basis and technical support for the biological control of the disease.

Cornus officinalis Sieb. et Zucc., belonging to the family Cornaceae, is often used as an ornamental plant and is widely distributed in Shandong, Jiangsu, and Zhejiang provinces and other places in China. Since 2020, a new disease with high incidence has been found in Xuanwu Lake Park (32°04'34.53″N 118°48'42.06″E) in Nanjing, Jiangsu Province, China. The symptoms began as small brown lesions formed along the leaf tips, which gradually expanded and became dark brown with a light brown border. A survey of C. officinalis trees in Xuanwu Lake Park showed that approximately 90% of thirty trees were infected, which decreased the ornamental value of C. officinalis. Pieces of leaf tissue (3 to 4 mm²) from the lesion margins were surface sterilized with 75% ethanol for 30 s and 1% NaClO for 90 s. Subsequently, the tissues were rinsed with sterile H2O, placed on potato dextrose agar (PDA) medium and incubated at 25℃ for 5 days. The same fungus was isolated in 90% of the tissues. Pure cultures were obtained by monosporic isolation. A representative isolate, SZY 2-2, was used for morphological and molecular characterization. The colonies were initially white, gradually turning gray green to black with copious gray aerial mycelium after 1 week in culture. Conidia were one-celled, hyaline, smooth, and fusoid to ellipsoid. Conidia measurements were 23.6±1.9×7.2±0.56 μm (n = 50). The morphology of SZY 2-2 matched the description of Botryosphaeria dothidea (Slippers et al. 2004). To verify species identity, the partial sequences of the internal transcribed spacer (ITS) region, translation elongation factor 1 alpha (EF1-a) gene, and beta-tubulin gene (TUB), were amplified from isolate SZY 2-2 with primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Koho 1999), and βt2a/βt2b (Glass and Donaldson 1995), respectively. The sequences were deposited in GenBank (ON171471 for ITS, ON185540 for EF1-a, and ON185541 for TUB). A BLAST search of GenBank showed that ITS, EF1-a and TUB sequences of SZY 2-2 were similar to those of B. dothidea MN633360 (identity=517/517 bp; 100%), MK783294 (identity=299/299 bp; 100%), and KF005081 (identity=461/461 bp; 100%), respectively. The morphological and molecular results identified the isolate as B. dothidea (Zhai et al. 2014). To fulfill Koch's postulates, a pathogenicity test was conducted using three C. officinalis plants. Five leaves from each tree were wounded and inoculated with mycelial plugs (about 4 mm in diameter) of B. dothidea from a 5-day-old culture grown on PDA, and inoculation with sterile PDA plugs on different leaves of the same tree served as negative controls. The leaves were enclosed in plastic bag along with the branches with a wet cotton ball inside. Sterile H2O2 was sprayed into the plastic bags to keep moisture conditions.Five days later, all inoculated points showed lesions similar to those previously observed in the field, whereas controls were asymptomatic. The pathogen was successfully reisolated from the inoculated symptomatic parts on PDA and had morphology as characterized before, thus fulfilling Koch's postulates. B. dothidea is known as a ubiquitous fungus and operates as both an endophyte and an opportunistic pathogen of trees (Slippers and Wingfield 2007, Zhao et al 2020). Stress factors that predispose trees to disease expression by B. dothidea include drought, defoliation (Theodore et al. 1997), competition, and physical damage (Slippers and Wingfield 2007). This is consistent with the occurrence of the disease in September and association of B. dothidea with the presence of wounds. More investigation is needed to determine the relationship between possible endophytic growth of B. dothidea on C. officinalis and the leaf blight found in Jiangsu Province.

Verticillium dahliae is one of the most destructive fungal pathogens, causing substantial economic losses in agriculture and forestry. The use of plant growth-promoting rhizobacteria (PGPR) is an effective and environmentally friendly strategy for controlling diseases caused by V. dahliae . In this study, 90 mm in diameter Petri plates were used to test the effect of volatile organic compounds (VOCs) produced by different concentrations of Pseudomonas aurantiaca ST-TJ4 cells suspension on V. dahliae mycelia radial growth and biomass. The mycelial morphology was observed by using scanning electron microscopy. The conidia germination and microsclerotia formation of V. dahliae were evaluated. The VOCs with antifungal activity were collected by headspace solid-phase microextraction (SPME), and their components were analyzed by gas chromatography-mass spectrometry (GC-MS). The VOCs produced by strain ST-TJ4 significantly inhibited the growth of mycelium of V. dahliae . The morphology of the hyphae was rough and wrinkled when exposed to VOCs. The VOCs of strain ST-TJ4 have a significant inhibitory effect on V. dahliae conidia germination and microsclerotia formation. At the same time, the VOCs also reduce the expression of genes related to melanin synthesis in V. dahliae . In particular, the expression of the hydrophobin gene ( VDAG - 02273 ) was down-regulated the most, about 67-fold. The VOCs effectively alleviate the severity of cotton root disease. In the volatile profile of strain ST-TJ4, 2-undecanone and 1-nonanol assayed in the range 10-200 µL per plate revealed a significant inhibitory effect on V. dahliae mycelial radial growth. These compounds may be useful to devise new control strategies for control of Verticillium wilt disease caused by V. dahliae .

Bursaphelenchus xylophilus is the most economically important species of migratory plant-parasitic nematodes (PPNs) and causes severe damage to forestry in China. The successful infection of B. xylophilus relies on the secretion of a repertoire of effector proteins. The effectors, which suppress the host pine immune response, are key to the facilitation of B. xylophilus parasitism. An exhaustive list of candidate effectors of B. xylophilus was predicted, but not all have been identified and characterized. Here, an effector, named BxSCD3, has been implicated in the suppression of host immunity. BxSCD3 could suppress pathogen-associated molecular patterns (PAMPs) PsXEG1- and INF1-triggered cell death when it was secreted into the intracellular space in Nicotiana benthamiana. BxSCD3 was highly up-regulated in the early infection stages of B. xylophilus . BxSCD3 does not affect B. xylophilus reproduction, either at the mycophagous stage or the phytophagous stage, but it contributes to the virulence of B. xylophilus. Moreover, BxSCD3 significantly influenced the relative expression levels of defense-related (PR) genes PtPR-3 and PtPR-6 in P inus thunbergii in the early infection stage. These results suggest that BxSCD3 is an important toxic factor and plays a key role in the interaction between B. xylophilus and host pine.

Ectomycorrhizal fungi (EMFs) form symbioses with plant roots to promote nutrient uptake by plants but it is controversial as to whether they induce disease resistance in plants. Here, we inoculated pine seedlings with Sphaeropsis sapinea , which was presymbiotic with the EMF Hymenochaete sp. Rl, and the mycorrhizal helper bacterium (MHB) Bacillus pumilus HR10, which promotes the formation of Pinus thunbergia - Hymenochaete sp. Rl mycorrhizae. The results showed that inoculation with Hymenochaete sp. Rl, B. pumilus HR10, and the consortium significantly reduced pine shoot blight disease caused by S. sapinea . After inoculation with pathogenic fungi, callose deposition was significantly increased in needles of pine seedlings inoculated with Hymenochaete sp. Rl, B. pumilus HR10, and the consortium, together with an increase in enzymatic and nonenzymatic systemic antioxidant activity as well as early priming for upregulated expression of PR3 and PR5 genes. Our findings suggest that ectomycorrhizal colonization enhances the resistance of pine seedlings to Sphaeropsis shoot blight by triggering a systemic defense response and that interactions between EMFs and MHBs are essential for mycorrhizal-induced disease resistance.

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., (© 2022. The Author(s).)

Background: The pine wilt disease (PWD) caused by Bursaphelenchus xylophilus is a devastating forest disease and its pathogenesis remains unclear. Secreted enzymes and proteins are important pathogenicity determinants and Bx-FAR-1 is an important pathogenic protein involved in the interaction between pine and B. xylophilus. However, the function of the Bx-FAR-1 protein in monitoring and prevention PWD remains unknown., Results: We found a small peptide of B. xylophilus effector Bx-FAR-1 is sufficient for immunosuppression function in Nicotiana benthamiana. Transient expression of Bx-FAR-1 in N. benthamiana revealed that nuclear localization is required for its function. The results of the ligand binding test showed that Bx-FAR-1 protein had the ability to bind fatty acid and retinol. We demonstrated that Bx-FAR-1 targeted to the nuclei of Pinus thunbergii using the polyclonal antibody by immunologic approach. The content of jasmonic acid (JA) was significantly increased in P. thunbergii infected with B. xylophilus when Bx-FAR-1 was silenced. We identified an F-box protein as the host target of Bx-FAR-1 by yeast two-hybrid and co-immunoprecipitation. Moreover, we found that Pt-F-box-1 was up-regulated during B. xylophilus infection and the expression of Pt-F-box-1 was increased in Bx-FAR-1 double-stranded RNA (dsRNA)-treated host pines., Conclusion: This study illustrated that Bx-FAR-1 might mediate the JA pathway to destroy the immune system of P. thunbergii, indicating that PWN likely secretes effectors to facilitate parasitism and promote infection, which could better reveal the pathogenesis mechanisms of B. xylophilus and would be beneficial for developing disease control strategies., (© 2022 Society of Chemical Industry.)

Background: Bursaphelenchus xylophilus is the causal agent of pine wilt disease (PWD) that has caused enormous ecological and economic losses in China. The mechanism in the interaction between nematodes and pine remains unclear. Plant parasitic nematodes (PPNs) secrete effectors into host plant tissues. However, it is poorly studied that role of effector in the infection of pine wood nematode (PWN)., Results: We cloned, characterized and functionally validated the B. xylophilus effector BxML1, containing an MD-2-related lipid-recognition (ML) domain. This protein inhibits immune responses triggered by the molecular pattern BxCDP1 of B. xylophilus. An insitu hybridization assay demonstrated that BxML1 was expressed mainly in the dorsal glands and intestine of B. xylophilus. Subcellular localization analysis showed the presence of BxML1 in the cytoplasm and nucleus. Furthermore, number of B. xylophilus and morbidity of pine were significantly reduced in Pinus thunbergii infected with B. xylophilus when BxML was silenced. Using yeast two-hybrid (Y2H) and coimmunoprecipitation (CoIP) assays, we found that the BxML1 interacts with cyclophilin protein PtCyP1 in P. thunbergii., Conclusions: This study illustrated that BxML1 plays a critical role in the B. xylophilus-plant interaction and virulence of B. xylophilus., (© 2022. The Author(s).)

Mycorrhizal helper bacteria (MHB) play an important role in mediating mycorrhizal symbiosis, which improves the growth and nutrient uptake of plants. This study examined the growth-promoting effects and mechanisms of pine growth after inoculation with the MHB Bacillus pumilus HR10 and/or Hymenochaete sp. Rl. The effect of B. pumilus HR10 on Hymenochaete sp. Rl growth, enzyme activity and gene expression related to mycorrhiza formation were determined. The growth, root activity, nitrogen, phosphorus, and potassium content and chlorophyll fluorescence activity of Pinus thunbergii and the mycorrhizal colonization intensity of Hymenochaete sp. Rl-inoculated pine seedlings after inoculation with B. pumilus HR10 were also evaluated. The results showed that B. pumilus HR10 promoted growth, regulated the expression of mycorrhizal-related genes and affected the β-1,3-glucanase activity of Hymenochaete sp. Rl. The mycorrhizal colonization intensity of pine seedlings co-inoculated with B. pumilus HR10 and Hymenochaete sp. Rl was 1.58-fold higher than seedlings inoculated with only Hymenochaete sp. Rl. Inoculation with B. pumilus HR10 and/or Hymenochaete sp. Rl increased lateral root number and root activity of pine seedlings and chlorophyll fluorescence activity of pine needles compared with the control. Bacillus pumilus HR10 facilitated nutrient uptake by enhancing the mycorrhizal proliferation of pine and induced greater photosynthesis and root activity of pine seedlings, which confirms its role as an outstanding plant-growth-promoting rhizobacterium. These findings improve our understanding of the mechanism of B. pumilus HR10 promotion of mycorrhizal symbiosis., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Genomic information for bacteria within the genus Rahnella remains limited. Rahnella sp. JZ-GX1 was previously isolated from the Pinus massoniana rhizosphere in China and shows potential as a plant growth-promoting (PGP) bacterium. In the present work, we combined the GridION Nanopore ONT and Illumina sequencing platforms to obtain the complete genome sequence of strain JZ-GX1, and the application effects of the strain in natural field environment was assessed. The whole genome of Rahnella sp. JZ-GX1 comprised a single circular chromosome (5,472,828 bp, G + C content of 53.53%) with 4,483 protein-coding sequences, 22 rRNAs, and 77 tRNAs. Based on whole genome phylogenetic and average nucleotide identity (ANI) analysis, the JZ-GX1 strain was reidentified as R. victoriana . Genes related to indole-3-acetic acid (IAA), phosphorus solubilization, nitrogen fixation, siderophores, acetoin, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, gamma-aminobutyric acid (GABA) production, spermidine and volatile organic compounds (VOCs) biosynthesis were present in the genome of strain JZ-GX1. In addition, these functions were also confirmed by in vitro experiments. Importantly, compared to uninoculated control plants, Pyrus serotina , Malus spectabilis , Populus euramericana (Dode) Guinier cv. "San Martino" (I-72 poplar) and Pinus elliottii plants inoculated with strain JZ-GX1 showed increased heights and ground diameters. These findings improve our understanding of R. victoriana JZ-GX1 as a potential biofertilizer in agriculture., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kong, Wang, Zuo and Wu.)

There are complex interactions between mycorrhizal helper bacteria (MHBs) and ectomycorrhizal (ECM) fungi, with MHBs promoting mycorrhizal synthesis and ECM fungi regulating plant rhizobacterial colonization, diversity, and function. In this study, to investigate whether the ECM fungus Hymenochaete sp. Rl affects the survival and colonization of the MHB strain Bacillus pumilus HR10 in the rhizosphere, the biomass of B. pumilus HR10 was measured in the rhizosphere and mycorrhizosphere. In addition, extracts of Hymenochaete sp. Rl and Pinus thunbergii were evaluated for their effect on B. pumilus HR10 colonization (growth, sporulation, biofilm formation, extracellular polysaccharide and extracellular protein contents, flagellar motility, and expression of colonization-related genes). The results showed that inoculation of Hymenochaete sp. Rl significantly increased the biomass of B. pumilus HR10 in the rhizosphere; however, while extracts of Hymenochaete sp. Rl and P. thunbergii did not affect the biomass or spore formation of HR10, they did affect its biofilm formation, extracellular polysaccharide and extracellular protein production, and flagellar motility. Furthermore, the addition of symbiont extracts affected the expression of chemotaxis-related genes in HR10. When the extracts were added separately, the expression of srf genes in HR10 increased; when the extracts were added simultaneously, the expression of the flagellin gene fliG in HR10 increased, but there was no significant effect on the expression of srf genes, consistent with the results on biofilm production. Thus, Hymenochaete sp. Rl and P. thunbergii roots had a positive effect on colonization by B. pumilus HR10 at the rhizosphere level through their secretions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Kong, Zhu, Dai and Wu.)

The MYB-CC family is a subtype within the MYB superfamily. This family contains an MYB domain and a predicted coiled-coil (CC) domain. Several MYB-CC transcription factors are involved in the plant's adaptability to low phosphate (Pi) stress. We identified 30, 34, and 55 MYB-CC genes in Brassica rapa , Brassica oleracea , and Brassica napus , respectively. The MYB-CC genes were divided into nine groups based on phylogenetic analysis. The analysis of the chromosome distribution and gene structure revealed that most MYB-CC genes retained the same relative position on the chromosomes and had similar gene structures during allotetraploidy. Evolutionary analysis showed that the ancestral whole-genome triplication (WGT) and the recent allopolyploidy are critical for the expansion of the MYB-CC gene family. The expression patterns of MYB-CC genes were found to be diverse in different tissues of the three Brassica species. Furthermore, the gene expression analysis under low Pi stress revealed that MYB-CC genes may be related to low Pi stress responses. These results may increase our understanding of MYB-CC gene family diversification and provide the basis for further analysis of the specific functions of MYB-CC genes in Brassica species., Competing Interests: The authors declare that they have no competing interests., (© 2022 Gu et al.)

Rhizosphere fungi have the beneficial functions of promoting plant growth and protecting plants from pests and pathogens. In our preliminary study, rhizosphere fungus JP-NJ4 was obtained from the soil rhizosphere of Pinus massoniana and selected for further analyses to confirm its functions of phosphate solubilization and plant growth promotion. In order to comprehensively investigate the function of this strain, it is necessary to ascertain its taxonomic position. With the help of genealogical concordance phylogenetic species recognition (GCPSR) using five genes/regions (ITS, BenA , CaM , RPB1 , and RPB2 ) as well as macro-morphological and micro-morphological characters, we accurately determined the classification status of strain JP-NJ4. The concatenated phylogenies of five (or four) gene regions and single gene phylogenetic trees (ITS, BenA , CaM , RPB1 , and RPB2 genes) all show that strain JP-NJ4 clustered together with Talaromyces brevis and Talaromyces liani , but differ markedly in the genetic distance (in BenA gene) from type strain and multiple collections of T . brevis and T . liani . The morphology of JP-NJ4 largely matches the characteristics of genes Talaromyces , and the rich and specific morphological information provided by its colonies was different from that of T. brevis and T. liani . In addition, strain JP-NJ4 could produce reduced conidiophores consisting of solitary phialides. From molecular and phenotypic data, strain JP-NJ4 was identified as a putative novel Talaromyces fungal species, designated T. nanjingensis .

The plant-parasitic nematode Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), causes enormous economic loss every year. Currently, little is known about the pathogenic mechanisms of PWD. Several effectors have been identified in B. xylophilus, but their functions and host targets have yet to be elucidated. Here, we demonstrated that BxSCD1 suppresses cell death and inhibits B. xylophilus PAMP BxCDP1-triggered immunity in Nicotiana benthamiana and Pinus thunbergii. BxSCD1 was transcriptionally upregulated in the early stage of B. xylophilus infection. In situ hybridization experiments showed that BxSCD1 was specifically expressed in the dorsal glands and intestine. Cysteine residues are essential for the function of BxSCD1. Transient expression of BxSCD1 in N. benthamiana revealed that it was primarily targeted to the cytoplasm and nucleus. The morbidity was significantly reduced in P. thunbergii infected with B. xylophilus when BxSCD1 was silenced. We identified 1-aminocyclopropane-1-carboxylate oxidase 1, the actual ethylene-forming enzyme, as a host target of BxSCD1 by yeast two-hybrid and coimmunoprecipitation. Overall, this study illustrated that BxSCD1 played a critical role in the B. xylophilus-plant interaction., (© 2021 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Salt stress is one of the major abiotic stresses that affects plant growth and development. The use of plant growth-promoting rhizobacteria to mitigcate salt stress damage in plants is an important way to promote crop growth under salt stress conditions. Rahnella aquatilis JZ-GX1 is a plant growth-promoting rhizobacterial strain, but it is not clear whether it can improve the salt tolerance of plants, and in particular, the role of volatile substances in plant salt tolerance is unknown. We investigated the effects of volatile organic compounds (VOCs) from JZ-GX1 on the growth performance, osmotic substances, ionic balance and antioxidant enzyme activities of acacia seedlings treated with 0 and 100mm NaCl and explored the VOCs associated with the JZ-GX1 strain. The results showed that compared to untreated seedlings, seedlings exposed to plant growth-promoting rhizobacterium JZ-GX1 via direct contact with plant roots under salt stress conditions exhibited increases in fresh weight, lateral root number and primary root length equal to approximately 155.1, 95.4, and 71.3%, respectively. Robinia pseudoacacia seedlings exposed to VOCs of the JZ-GX1 strain showed increases in biomass, soil and plant analyser development values and lateral root numbers equal to 132.1, 101.6, and 166.7%, respectively. Additionally, decreases in malondialdehyde, superoxide anion (O 2 - ) and hydrogen peroxide (H 2 O 2 ) contents and increases in proline contents and superoxide dismutase, peroxidase and glutathione reductase activities were observed in acacia leaves. Importantly, the sodium-potassium ratios in the roots, stems, and leaves of acacia exposed to VOCs of the JZ-GX1 strain were significantly lower than those in the control samples, and this change in ion homeostasis was consistent with the upregulated expression of the (Na + , K + )/H + reverse cotransporter RpNHX1 in plant roots. Through GC-MS and creatine chromatography, we also found that 2,3-butanediol in the volatile gases of the JZ-GX1 strain was one of the important signaling substances for improving the salt tolerance of plants. The results showed that R. aquatilis JZ-GX1 can promote the growth and yield of R. pseudoacacia under normal and salt stress conditions. JZ-GX1 VOCs have good potential as protectants for improving the salt tolerance of plants, opening a window of opportunity for their application in salinized soils., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Kong, Wu and Zhang.)

Phytase plays an important role in crop seed germination and plant growth. In order to fully understand the plant growth-promoting mechanism by Rahnella   aquatilis JZ-GX1, the effect of this strain on germination of maize seeds was determined in vitro, and the colonization of maize root by R. aquatilis JZ-GX1 was observed by scanning electron microscope. Different inoculum concentrations and Phytate-related soil properties were applied to investigate the effect of R. aquatilis JZ-GX1 on the growth of maize seedlings. The results showed that R. aquatilis JZ-GX1 could effectively secrete indole acetic acid and had significantly promoted seed germination and root length of maize. A large number of R. aquatilis JZ-GX1 cells colonized on the root surface, root hair and the root interior of maize. When the inoculation concentration was 10 7 cfu/mL and the insoluble organophosphorus compound phytate existed in the soil, the net photosynthetic rate, chlorophyll content, phytase activity secreted by roots, total phosphorus concentration and biomass accumulation of maize seedlings were the highest. In contrast, no significant effect of inoculation was found when the total P content was low or when inorganic P was sufficient in the soil. R. aquatilis JZ-GX1 promotes the growth of maize directly by secreting IAA and indirectly by secreting phytase. This work provides beneficial information for the development and application of R. aquatilis JZ-GX1 as a microbial fertilizer in the future.

Iron deficiency in soil has crucially restricted agricultural and forestry production. Volatile organic compounds (VOCs) produced by beneficial microorganisms have been proven to play an important role in inducing abiotic stress tolerance in plants. We investigated the effects of VOCs released by the rhizobacterium Rahnella aquatilis JZ-GX1 on the growth and root parameters of Arabidopsis thaliana under iron deficiency. The effect of the rhizobacterial VOCs on the gene expression in iron uptake and hormone signaling pathways were detected by RT-qPCR. Finally, the VOCs of the JZ-GX1 strain that could promote plant growth under iron deficiency stress were screened. The results showed that the JZ-GX1 strain could induce A. thaliana tolerance to iron deficiency stress by promoting the development of lateral roots and root hairs and increasing the activities of H + ATPase and Fe 3+ reductase. In addition, the AHA2 , FRO2 , and IRT1 genes of A. thaliana exposed to JZ-GX1-emitted VOCs were upregulated 25-, 1. 81-, and 1.35-fold, respectively, and expression of the abscisic acid (ABA) synthesis gene NCED3 was upregulated on both the 3rd and 5th days. Organic compounds were analyzed in the headspace of JZ-GX1 cultures, 2-undecanone and 3-methyl-1-butanol were found to promote Medicago sativa and A. thaliana growth under iron-limited conditions. These results demonstrated that the VOCs of R. aquatilis JZ-GX1 have good potential in promoting iron absorption in plants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kong, Wang and Wu.)

Salinity is one of the strongest abiotic factors in nature and has harmful effects on plants and microorganisms. In recent years, the degree of soil salinization has become an increasingly serious problem, and the use of plant growth-promoting rhizobacteria has become an option to improve the stress resistance of plants. In the present study, the salt tolerance mechanism of the rhizosphere bacterium Rahnella aquatilis JZ-GX1 was investigated through scanning electron microscopy observations and analysis of growth characteristics, compatible solutes, ion distribution and gene expression. In addition, the effect of JZ-GX1 on plant germination and seedling growth was preliminarily assessed through germination experiments. R. aquatilis JZ-GX1 was tolerant to 0-9% NaCl and grew well at 3%. Strain JZ-GX1 promotes salt tolerance by stimulating the production of exopolysaccharides, and can secrete 60.6983 mg/L of exopolysaccharides under the high salt concentration of 9%. Furthermore, the accumulation of the compatible solute trehalose in cells as the NaCl concentration increased was shown to be the primary mechanism of resistance to high salt concentrations in JZ-GX1. Strain JZ-GX1 could still produce indole-3-acetic acid (IAA) and siderophores and dissolve inorganic phosphorus under salt stress, characteristics that promote the ability of plants to resist salt stress. When the salt concentration was 100 mmol/L, strain JZ-GX1 significantly improved the germination rate, germination potential, fresh weight, primary root length and stem length of tomato seeds by 10.52, 125.56, 50.00, 218.18, and 144.64%, respectively. Therefore, R. aquatilis JZ-GX1 is a moderately halophilic bacterium with good growth-promoting function that has potential for future development as a microbial agent and use in saline-alkali land resources., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Kong and Wu.)

Background: The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a devastating pathogen of many Pinus species in China. The aim of this study was to understand the interactive molecular mechanism of PWN and its host by comparing differentially expressed genes and candidate effectors from three transcriptomes of B. xylophilus at different infection stages., Results: In total, 62, 69 and 46 candidate effectors were identified in three transcriptomes (2.5 h postinfection, 6, 12 and 24 h postinoculation and 6 and 15 d postinfection, respectively). In addition to uncharacterized pioneers, other candidate effectors were involved in the degradation of host tissues, suppression of host defenses, targeting plant signaling pathways, feeding and detoxification, which helped B. xylophilus survive successfully in the host. Seven candidate effectors were identified in both our study and the B. xylophilus transcriptome at 2.5 h postinfection, and one candidate effector was identified in all three transcriptomes. These common candidate effectors were upregulated at infection stages, and one of them suppressed pathogen-associated molecular pattern (PAMP) PsXEG1-triggered cell death in Nicotiana benthamiana., Conclusions: The results indicated that B. xylophilus secreted various candidate effectors, and some of them continued to function throughout all infection stages. These various candidate effectors were important to B. xylophilus infection and survival, and they functioned in different ways (such as breaking down host cell walls, suppressing host defenses, promoting feeding efficiency, promoting detoxification and playing virulence functions). The present results provide valuable resources for in-depth research on the pathogenesis of B. xylophilus from the perspective of effectors.

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).

Chaenomeles sinensis is a shrub or small arbor of the genus Chaenomeles in Rosaceae, which is widely planted in China. It is a kind of garden ornamental plant and has high economic value. Since 2020, a leaf disease occurred on the foliage of C. sinensis at the campus of Nanjing Forestry University, Nanjing, China. After investigating, C. sinensis was found with leaf spot disease at a 100% infection rate, which causing gigantic ornamental loss. Leaf spots are round to irregular distributing on the leaves, in addition, the color of spots is brown. There are yellow halos on the edge of the lesion. Small leaf tissues (3 to 4 mm2) from lesion margins were surface sterilized with 75% ethanol for 30s and then rinsed with sterile dH2O for three times. Afterwards, placed on potato dextrose agar (PDA) at 25°C. Pure cultures were obtained by monosporic isolation, and a representative isolate (NJTJ.1) was obtained. When cultured on PDA, the colony of NJTJ.1 was white and cottony. On the reverse side, the color of colony nearly light yellow. The colony were placed in the liquid Carboxymethyl cellulose (CMC) medium. After culturing for 24h in a shaker at 25℃ and 150rmp/min, the spore liquid was taken by us. The conidia were one-celled, straight, hyaline, subcylindrical with rounded ends and measured 15.1 to 23.6× 5.4 to 7.9 µm (n =30). Appressoria were one-celled, brown, thick-walled, ellipsoidal, and measured 7.7 to 13.8 × 6.4 to 10.3 µm (n =30). The morphological characteristics of NJTJ.1 fitted with the description of the Colletotrichhum gloeosporioides complex (Weir et al., 2012). For accurate identification, the internal transcribed spacer (ITS), and the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT) and chitin synthase (CHS-1) were amplified with primers ITS1/ITS4, GDF/GDR, ACT-512F/ACT-783R, and CHS-79F/CHS-345R (Zhu et al, 2019), respectively. The sequences were deposited in GenBank [Accession Nos.MT984264, MW030495 and MW030496 to MW030497 for NJTJ.1]. A Blast search of GenBank showed that ITS, GAPDH, ACT and CHS-1 sequences of NJTJ.1 were 99%, 99%, 100% and 100% identical to those of C. gloeosporioides (MH571757.1 ,KY995355.1 , MN058143.1 and MN313581.1). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate NJTJ.1 clustered in the C. gloeosporioides clade with 99% bootstrap support. The pathogenicity of the NJTJ.1 was verified both on detached and living leaves. The detached leaves were inoculated with 5-mm mycelial plugs cut from the edge of 6-day old cultures on PDA and 20 μL of spore suspension (106 conidia/mL) and each treatment had 5 replicates. Controls were treated with sterile dH2O. The inocula were placed at a distance of 2 to 3 cm on the leaves which were wounded with a sterile needle. All of them were placed in 20-cm dishes on wet filter paper at 25°C. After 5 days, all the inoculated points showed lesions which were similar to those outdoor observed. Whereas, controls were asymptomatic.At the same time, the plugs of C. gloeosporioides were inoculated on living leaves.After 7 days, the leaves which were inoculated also appeared lesions. This is the first report of C. gloeosporioides causing leaf blotch on Chaenomeles sinensis in China. These data will help develop effective strategies for managing this disease.

Background: Immunological disease-related chronic cerebrospinal venous insufficiency (CCSVI) is rarely reported. This study aimed to analyze clinical characteristics, inflammation, and coagulation status in patients with immunological disease-related CCSVI., Methods: Patients with CCSVI were enrolled from 2017 to 2019 and divided into three cohorts based on their immunological disease backgrounds, including groups with confirmed autoimmune disease, with suspected/subclinical autoimmune disease, and with non-immunological etiology. Immunological, inflammatory, and thrombophilia biomarker assay in blood samples were obtained. Mann-Whitney U test or Fisher's exact test was used to compare continuous variables or categorical variables between the CCSVI patients with or without the immunological etiology. Spearman's correlation analysis was conducted among age, baseline neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), interleukin-6 (IL-6), C-reactive protein (CRP), and neuron-specific enolase (NSE) in the three groups., Results: A total of 255 consecutive patients with CCSVI were enrolled, including three subgroups: CCSVI with confirmed autoimmune disease (n=41), CCSVI with suspected/subclinical autoimmune disease (n=116) and CCSVI with non-immunological etiology (n=98). In the first subgroup, a series of 41 cases was confirmed with eight different autoimmune diseases including antiphospholipid syndrome (n=18), Sjögren's syndrome (n=8), immunoglobulin G4-related disease (n=7), Behçet's disease (n=2), autoimmune hepatitis (n=2), Wegener's granulomatosis (n=2), systemic sclerosis (n=1) and AQP4 antibody-positive neuromyelitis optica spectrum disorder (n=1). Groups with immunological etiology did not show a higher incidence of thrombophilia or increased pro-inflammatory biomarkers (e.g., neutrophil, IL-6). However, patients with non-immunological etiology had a higher baseline level of CRP. Additionally, baseline PLR was moderately correlated to NLR and CRP in CCSVI patients with non-immunological etiology and suspected/subclinical autoimmune disease., Conclusions: The formation of CCSVI may be based on the inflammatory process, facilitated by multiple risk factors, among which medical history of immunological diseases may play a significant role due to the intricate relationship between inflammation and coagulation. Moreover, CCSVI may also cause an independent inflammatory injury in venous walls, leading to focal stenosis or thrombus, without attacks from autoimmune antibodies., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-4201). The authors have no conflicts of interest to declare., (2021 Annals of Translational Medicine. All rights reserved.)

Fungal esophagitis is a common infectious disease, although the pathogenic clinical characteristics remain incompletely clear, especially in South China. The goal of this study was to investigate the pathogenic clinical characteristics of fungal esophagitis and the efficacy of different therapeutic strategies at a tertiary hospital in South China. A retrospective study was conducted from January 2007 to December 2017. Data from 113,390 patients who were treated in the endoscopic unit were retrieved and analyzed. To further understand the pathogen and risk factors for fungal esophagitis, we performed a case-control analysis of 101 patients and 202 controls. Of the 113,390 patients, 932 (0.82%) were positive. The annual detection rate ranged from 0.345% to 1.106%, showing an initially increasing and subsequently decreasing trend. The patients' median age was 49 years (range from 8 to 85), and most were men (615/932, 65.99%). Candida albicans was found in samples collected from 36 patients, without any drug-resistant strains. Age ( P = 0.018), malignancy (OR = 4.031, 95% CI: 1.562~10.407), cigarette smoking (OR = 3.017, 95% CI: 1.645~5.533), and the use of antibiotics (OR = 2.178, 95% CI: 1.078~4.400) or immunosuppressants (OR = 6.525, 95% CI: 1.089~39.105) were independently associated with esophageal candidiasis. Fluconazole had a better curative effect than nystatin (OR = 4.047, 95% CI: 1.282~12.772) or simple observation (OR = 8.91, 95% CI: 2.61~30.49). In conclusion, fungal esophagitis primarily affects men and elderly individuals; it develops in the setting of malignancy, smoking, and certain previous medication use. Candida albicans is the most common pathogen and is sensitive to antifungal agents. Fluconazole has a good therapeutic effect., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2021 Shu-Kai Zhan et al.)

An electrochemical biosensor based on a water-stable one-dimensional double-chain Cu(II) metal-organic framework (Cu-MOF) directly was constructed for efficiently recognizing l-tyrosine (l-Tyr) in biomimic environments. Cu-MOF: {[Cu(bpe)(fdc) (H 2 O)(DMF)]·0.5H 2 O} n (bpe = 1,2-di(4-pyridyl)ethylene, H 2 fdc = 2,5-furandicarboxylic acid, namely, Cu-1 ) was synthesized by a hydrothermal method. It was characterized by IR, scanning electron microscopy, atomic force microscopy, and PXRD techniques. Cu-1 exhibited extreme solvent and thermal stability as well as excellent electroconductive character. It was coated on a glassy carbon electrode (GCE) surface to prepare an electrochemical biosensor ( Cu-1 /GCE) which showed preferable biosensing ability toward l-Tyr. This Cu-MOF electrochemical biosensor showed simple operation and high sensitivity toward l-Tyr in the concentration range from 0.01 to 0.09 mM. The detection limit is 5.822 μM. Furthermore, Cu-1 /GCE showed extremely excellent selectivity to l-Tyr in a biomimic environment with several amino acid interferents. This new strategy exhibits great potential applications for designing MOFs with excellent electrochemical activity.

Pecan (Carya illinoinensis) is important and widely planted nut tree species in Anhui province in China. In a pecan orchard in Anhui, China, 54% of the 1-year-old container-grown seedlings in the nursery developed leaf spots in September 2019. Initially, the brown spots appeared on the leaves. Later, the spots expanded to become brown circles surrounded by a dark brown border. Under severe infection, defoliation occurred and black acervuli were observed on symptomatic leaves. Disease symptoms were not observed on the fruits. To isolate the pathogen, leaf sections (3 to 4 mm) were excised from the margin of the diseased leaf tissues, surface sterilized in 75% alcohol for 30 s and then in 0.1% mercuric chloride for 30s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25 °C in the darkness. Pure cultures were obtained by monosporic isolation. The colony of a representative isolate, CZ-4, growing on PDA was circular, white, and cottony, and the surface undulate and pale luteous on PDA. The reverse similar in color. The conidial masses were black and appeared over PDA plates after 14 days. Conidia (15.41-29.48×4.15-7.54µm) (n=50) were fusiform to ellipsoid and four-septate (one basal and one apical cell hyaline, and three brown median cells), with two to three apical appendages. According to colony and conidia morphology, the isolates were identified as Neopestalotiopsis sp. (Maharachchikumbura et al. 2014). Genomic DNA was extracted from single conidial cultures of a representative isolate CZ-4, and the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1), β-tubulin (TUB) genes were amplified with the primers described by Wang et al. (2019). The obtained sequences showed 95-99% similarity with those from Neopestalotiopsis chrysea accessions in GenBank. The sequences from this isolate were deposited in GenBank under the following accession numbers: ITS, MT459336; TEF1, MT469880; TUB, MT469881. A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate CZ-4 clustered in the N. chrysea clade with 98% bootstrap support. To test pathogenicity, ten detached healthy leaves and ten one-year-old Carya illinoinensis plants were inoculated with the same pathogens by spraying 50µl of a conidial suspension (1×106 conidia/ml) on both sides of leaves.As a control treatment, ten additional detached leaves and potted seedlings were inoculated with 50µl sterile water. All plants were covered with clear polyethylene bags and incubated in a greenhouse (Center of Co-Innovation, Institute of Forestry) at 23 ± 5 °C, 80% relative humidity, and a 12-h light/dark cycle.The experiment was repeated three times. Seven days after inoculation, the symptoms were similar to those on the original infected plants, whereas the control leaves remained symptomless. N. chrysea was re-isolated from the lesions, morphologically identified, confirming Koch's postulates. To our knowledge, this is the first report of N. chrysea associated with leaf spot disease on C. illinoinensis. This study provides the foundation to further investigate the biology, epidemiology, and management of this disease.

Background: Autophagy plays an important role in eukaryotes. We investigated its role in the pine wood nematode (PWN), Bursaphelenchus xylophilus, the causative agent of pine wilt disease (PWD), to find promising control strategies against PWD., Results: We analysed the expression levels of PtRBOH1 and PtRBOH2, which regulate reactive oxygen species (ROS) metabolism, in Pinus thunbergii and the expression of three autophagy genes, BxATG5, BxATG9 and BxATG16, in PWN by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and measured the content of H 2 O 2 , the main product of ROS metabolism, in pine stem. There was a correlation between the expression of autophagy genes in PWN and pine ROS metabolism during early infection. We also found that oxidative stress induces autophagy in PWN according to qRT-PCR, transmission electron microscopy and Western blot analyses. Inhibition of autophagy by 3-methyladenine or silencing of the autophagy genes BxATG9 and BxATG16 in PWN showed that autophagy is essential for feeding, fecundity, egg hatching and survival of PWN under oxidative stress, confirming the importance of autophagy in the antioxidant defences of PWN. Similarly, we demonstrated that autophagy contributes to the virulence of PWN. Moreover, PWN likely ameliorates oxidative damage by enhancing the activities of the peroxidase and catalase antioxidant pathways when autophagy is inhibited., Conclusion: Autophagy contributes to resistance to the oxidative stress induced by pine ROS metabolism, thus promoting infection by PWN. Our findings clarify the defence mechanisms of PWN and the pathogenesis of PWD, and provide promising hints for control of PWD by blocking autophagy. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

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 Fe 2+ , Mg 2+ , Ca 2+ , 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.

The migratory plant-parasitic nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease, which causes serious damage to pine forests in China. Plant immunity plays an important role in plant resistance to multiple pathogens. Activation of the plant immune system is generally determined by immune receptors, including plant pattern recognition receptors, which mediate pattern recognition. However, little is known about molecular pattern recognition in the interaction between pines and B. xylophilus. Based on the B. xylophilus transcriptome at the early stages of infection and Agrobacterium tumefaciens-mediated transient expression and infiltration of recombinant proteins produced by Pichia pastoris in many plant species, a novel molecular pattern (BxCDP1) was characterized in B. xylophilus. We found that BxCDP1 was highly up-regulated at the early infection stages of B. xylophilus, and was similar to a protein in Pararhizobium haloflavum. BxCDP1 triggered cell death in Nicotiana benthamiana when secreted into the apoplast, and this effect was dependent on brassinosteroid-insensitive 1-associated kinase 1, but independent of suppressor of BIR1-1. BxCDP1 also exhibited cell death-inducing activity in pine, Arabidopsis, tomato, pepper, and lettuce. BxCDP1 triggered reactive oxygen species production and the expression of PAMP-triggered immunity marker genes (NbAcre31, NbPTI5, and NbCyp71D20) in N. benthamiana. It also induced the expression of pathogenesis-related genes (PtPR-3, PtPR-4, and PtPR-5) in Pinus thunbergii. These results suggest that as a new B. xylophilus molecular pattern, BxCDP1 can not only be recognized by many plant species, but also triggers innate immunity in N. benthamiana and defence responses of P. thunbergii., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

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.