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Global citrus production has been severely affected by citrus Huanglongbing (HLB) disease, caused by Candidatus Liberibacter asiaticus (Clas), and the development of effective control methods are crucial. This study employed antimicrobial lipopeptide and phytohormone complex powder (L1) prepared from the fermentation broth of the endophytic plant growth promoting bacterium (PGPB) of Bacillus amyloliquefaciens strain MG-2 to treat Candidatus Liberibacter asiaticus (CLas)-infected ‘Citrus reticulata ‘Chun Jian’ plants. Real-time fluorescence quantitative polymerase chain reaction (qPCR) and PCR were employed for disease detection. The results revealed that after 15 spray-drench treatments with L1 solution, the HLB infection rate decreased from 100 to 50%, the bacterial titer decreased by 51.9% compared with a 27.9% decrease in the control group. L1 treatment triggered the production of reactive oxygen species, increased lignin content, and increased defense enzyme activities (p 2, p

Background: An ideal animal model to study SARS-coronavirus 2 (SARS-CoV-2) pathogenesis and evaluate therapies and vaccines should reproduce SARS-CoV-2 infection and recapitulate lung disease like those seen in humans. The angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS-CoV-2, but mice are resistant to the infection because their ACE2 is incompatible with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein . Methods: SARS-CoV-2 was passaged in BALB/c mice to obtain mouse-adapted virus strain. Complete genome deep sequencing of different generations of viruses was performed to characterize the dynamics of the adaptive mutations in SARS-CoV-2. Indirect immunofluorescence analysis and Biolayer interferometry experiments determined the binding affinity of mouse-adapted SARS-CoV-2 WBP-1 RBD to mouse ACE2 and human ACE2. Finally, we tested whether TLR7/8 agonist Resiquimod (R848) could also inhibit the replication of WBP-1 in the mouse model. Findings: The mouse-adapted strain WBP-1 showed increased infectivity in BALB/c mice and led to severe interstitial pneumonia. We characterized the dynamics of the adaptive mutations in SARS-CoV-2 and demonstrated that Q493K and Q498H in RBD significantly increased its binding affinity towards mouse ACE2. Additionally, the study tentatively found that the TLR7/8 agonist Resiquimod was able to protect mice against WBP-1 challenge. Therefore, this mouse-adapted strain is a useful tool to investigate COVID-19 and develop new therapies. Interpretation: We found for the first time that the Q493K and Q498H mutations in the RBD of WBP-1 enhanced its interactive affinities with mACE2. The mouse-adapted SARS-CoV-2 provides a valuable tool for the evaluation of novel antiviral and vaccine strategies. This study also tentatively verified the antiviral activity of TLR7/8 agonist Resiquimod against SARS-CoV-2 in vitro and in vivo. Funding: This research was funded by the National Key Research and Development Program of China (2020YFC0845600) and Emergency Science and Technology Project of Hubei Province (2020FCA046) and Robert A. Welch Foundation (C-1565).

Citrus production is facing an unprecedented problem because of huanglongbing (HLB) disease. Presently, no effective HLB-easing method is available when citrus becomes infected. Guanosine 5′-monophosphate synthetase (GMPS) is a key protein in the de novo synthesis of guanine nucleotides. GMPS is used as an attractive target for developing agents that are effective against the patogen infection. In this research, homology modeling, structure-based virtual screening, and molecular docking were used to discover the new inhibitors against CLas GMPS. Enzyme assay showed that folic acid and AZD1152 showed high inhibition at micromole concentrations, with AZD1152 being the most potent molecule. The inhibition constant (Ki) value of folic acid and AZD1152 was 51.98 µM and 4.05 µM, respectively. These results suggested that folic acid and AZD1152 could be considered as promising candidates for the development of CLas agents.

Citrus huanglongbing (HLB) is a destructive disease caused by Candidatus Liberibacter species and is a serious global concern for the citrus industry. To date, there is no established strategy for control of this disease. Previously, Bacillus amyloliquefaciens GJ1 was screened as the biocontrol agent against HLB. In this study, two-year-old citrus infected by Ca. L. asiaticus were treated with B. amyloliquefaciens GJ1 solution via root irrigation. In these plants, after seven irrigation treatments, the results indicated that the photosynthetic parameters, chlorophyll content, resistance-associated enzyme content and the expression of defense-related genes were significantly higher than for the plants treated with the same volume water. The content of starch and soluble sugar were significantly lower, compared to the control treatment. The parallel reaction monitoring (PRM) results revealed that treatment with B. amyloliquefaciens GJ1 solution, the expression levels of 3 proteins with photosynthetic function were upregulated in citrus leaves. The accumulation of reactive oxygen species (ROS) in citrus leaves treated with B. amyloliquefaciens GJ1 flag22 was significantly higher than untreated plants and induced the defense-related gene expression in citrus. Finally, surfactin was identified from the fermentation broth of B. amyloliquefaciens GJ1 by high-performance liquid chromatography. These results indicate that B. amyloliquefaciens GJ1 may improve the immunity of citrus by increasing the photosynthesis and enhancing the expression of the resistance-related genes.

Citrus huanglongbing (HLB) is a destructive disease that causes significant damage to many citrus producing areas worldwide. To date, no strategy against this disease has been established. Inosine 5′-monophosphate dehydrogenase (IMPDH) plays crucial roles in the de novo synthesis of guanine nucleotides. This enzyme is used as a potential target to treat bacterial infection. In this study, the crystal structure of a deletion mutant of CLas IMPDHΔ98-201 in the apo form was determined. Eight known bioactive compounds were used as ligands for molecular docking. The results showed that bronopol and disulfiram bound to CLas IMPDHΔ98-201 with high affinity. These compounds were tested for their inhibition against CLas IMPDHΔ98-201 activity. Bronopol and disulfiram showed high inhibition at nanomolar concentrations, and bronopol was found to be the most potent molecule (Ki = 234 nM). The Ki value of disulfiram was 616 nM. These results suggest that bronopol and disulfiram can be considered potential candidate agents for the development of CLas inhibitors.