Fengycin produced by Bacillus subtilis XF-1 plays a major role in the biocontrol of Chinese cabbage clubroot via direct effect and defense stimulation.

Bacillus subtilis XF-1 is a well-investigated biocontrol agent against the biotrophic Plasmodiophora brassicae Woron., the causal agent of clubroot disease of cruciferous crops. The present study demonstrates that XF-1 could efficiently control clubroot disease via leaf spraying and provides an understanding of the biocontrol mechanisms. High-performance thin-layer chromatography (HTPLC) analysis indicated the presence of fengycin-type cyclopeptides in the supernatant. A ppsB deletion mutant of XF-1 resulted in no fengycin production, significantly reduced the lysis rate of testing spores in vitro and the primary infection rate of root hair in vivo, and decreased the protection value against clubroot disease under the greenhouse conditions. Confocal laser scanning microscopy proved that fengycin was not required for leaf internalization and root colonization. Moreover, the expression level of the ppsB gene in XF-1 was regulated by its cell density in root during interaction with P. brassicae. In addition, the ΔppsB mutant of XF-1 could not efficiently control disease because it led to a lower activation level of the jasmonic acid and salicylic acid signaling pathways in roots, which are necessary for the plant defense reaction upon pathogen invasion. Altogether, the present study provides a new understanding of specific cues in the interaction between B. subtilis and P. brassicae as well as insights into the application of B. subtilis in agriculture.

Complete Genome Sequence of Bacillus velezensis TH-1, a Candidate Biocontrol Bacterium from China.

Bacillus velezensis TH-1 is a plant growth-promoting rhizobacteria with biocontrol potential that was isolated from the rhizosphere of Sophora tonkinensis Radix. Our previous results showed that strain TH-1 demonstrated effective biocontrol activity against root rot of Sophora tonkinensis Radix and bacterial wilt of ginger. Currently, only a few whole-genome sequences of biocontrol strains isolated from the rhizosphere of medicinal plants are available. We report, here, the complete genome sequence of B. velezensis TH-1. The size of TH-1 genome is 3,929,846 bp that consists of 3,900 genes with a total GC content of 46.48%. The strain TH-1 genome has 3,661 coding genes, 86 transfer RNAs, 27 ribosomal RNAs, and 16 small RNAs. Moreover, we identified nine gene clusters coding for the biosynthesis of antimicrobial compounds. The genomic information of TH-1 will provide resources for the study of biological control mechanisms and plant-microbe interactions. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Corrigendum: Defeating Huanglongbing Pathogen Candidatus Liberibacter asiaticus With Indigenous Citrus Endophyte Bacillus subtilis L1-21.

[This corrects the article DOI: 10.3389/fpls.2021.789065.].

[Role of p22phox and NOX5 in autophagy and apoptosis of osteoblasts induced by hypoxia].

OBJECTIVE: To investigate the role of p22phox and NOX5 in autophagy and apoptosis of osteoblasts induced by hypoxia. METHODS: The skull tissue of newborn rats was cut into small pieces, and the osteoblasts were separated and purified by the tissue block adherent method and the differential adherent method. The first generation cells were harvested and identified by HE staining, Alizarin red staining, alkaline phosphatase (ALP) staining, and flow cytometry. A three-gas incubator was used to prepare a hypoxia model of osteoblasts. At 0, 3, 6, 12, and 24 hours of hypoxia, the expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ were detected by Western blot, and the level of reactive oxygen species (ROS) and cell apoptosis rate were detected by flow cytometry. And the time point of the highest level of ROS was selected as the hypoxia time point for subsequent experiments. The first generation osteoblasts were divided into normal group, si-p22phox hypoxia group, and si-NOX5 hypoxia group and subjected to corresponding transfection and hypoxia treatment. The inhibition efficiency of si-p22phox and si-NOX5 were detected by RT-PCR. Then the osteoblasts were divided into normal group, si-NC hypoxia group, si-p22phox hypoxia group, and si-NOX5 hypoxia group. After transfection and hypoxia treatment, Western blot was used to detect the expressions of p22phox, NOX5, autophagy-related proteins (LC3Ⅱ/Ⅰ, Beclin), and apoptosis-related proteins (Bcl-2, Bax), and flow cytometry was used to detect the cell apoptosis rate and level of ROS. The first generation osteoblasts were divided into a hypoxia group for 12 hours (hypoxia group) and a group that simultaneously inhibited si-p22phox and si-NOX5 and hypoxia for 12 hours (inhibition+hypoxia group). The expressions of Beclin and Bax were observed by immunofluorescence staining after the corresponding treatment. RESULTS: After identification, the isolated cells were osteoblasts. After hypoxia treatment, the relative expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ proteins and the apoptosis rate of osteoblasts gradually increased ( P<0.05), and the level of ROS also significantly increased ( P<0.05) and reached the peak value at 12 hours. The 12-hour hypoxia model was selected for subsequent experiments. Silencing the p22phox gene did not affect the expression of NOX5, and silencing the NOX5 gene did not affect the expression of p22phox. Compared with hypoxia treatment, the relative expressions of LC3Ⅱ/Ⅰ, Beclin, and Bax proteins after inhibiting the expression of p22phox or NOX5 gene significantly decreased ( P<0.05), the relative expression of Bcl-2 protein significantly increased ( P<0.05), the cell apoptosis rate and level of ROS also significantly decreased ( P<0.05). After silencing the expressions of p22phox and NOX5 genes at the same time, the immunofluorescence staining showed that the fluorescence of Beclin and Bax were weak. CONCLUSION: Inhibiting the expressions of p22phox and NOX5 genes can reduce the level of ROS in osteoblasts under hypoxia-induced conditions, and at the same time reduce autophagy and apoptosis, especially attenuate the excessive apoptosis of cells in the early to late stages, and strengthen the hypoxic osteoblasts proliferation.

Defeating Huanglongbing Pathogen Candidatus Liberibacter asiaticus With Indigenous Citrus Endophyte Bacillus subtilis L1-21.

Huanglongbing (HLB) has turned into a devastating botanical pandemic of citrus crops, caused by Candidatus Liberibacter asiaticus (CLas). However, until now the disease has remained incurable with very limited control strategies available. Restoration of the affected microbiomes in the diseased host through the introduction of an indigenous endophyte Bacillus subtilis L1-21 isolated from healthy citrus may provide an innovative approach for disease management. A novel half-leaf method was developed in vitro to test the efficacy of the endophyte L1-21 against CLas. Application of B. subtilis L1-21 at 104 colony forming unit (cfu ml-1) resulted in a 1,000-fold reduction in the CLas copies per gram of leaf midrib (107 to 104) in 4 days. In HLB-affected citrus orchards over a period of 2 years, the CLas incidence was reduced to < 3%, and CLas copies declined from 109 to 104 g-1 of diseased leaf midribs in the endophyte L1-21 treated trees. Reduction in disease incidence may corroborate a direct or an indirect biocontrol effect of the endophytes as red fluorescent protein-labeled B. subtilis L1-21 colonized and shared niche (phloem) with CLas. This is the first large-scale study for establishing a sustainable HLB control strategy through citrus endophytic microbiome restructuring using an indigenous endophyte.

[Expression and correlation analysis of hypoxia inducible factor 1α and autophagy related molecules in rat nucleus pulposus cells under hypoxia].

OBJECTIVE: To investigate the expression and correlation of hypoxia inducible factor 1α (HIF-1α) and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells under hypoxia in vitro. METHODS: The nucleus pulposus cells were extracted from the nucleus pulposus of healthy adult Sprague Dawley rats and passaged. The 3rd generation cells were identified by HE staining and collagenase type Ⅱ immunofluorescence staining and randomly divided into 4 groups. The cells in group A were cultured for 8 hours under normal oxygen condition (37℃, 5%CO 2, 20%O 2); the cells in group B were cultured for 8 hours under hypoxia condition (37℃, 5%CO 2, 1%O 2); the cells in group C were transfected with HIF-1α-small interfering RNA and cultured for 8 hours under hypoxia condition; and the cells in group D were cultured with autophagy inhibitor 3-MA for 8 hours under hypoxia condition. Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to detect the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in all groups. RESULTS: HE staining of the 3rd generation nucleus pulposus cells showed that the cytoplasm was light pink and the nucleus was blue black, and the collagenase type Ⅱ immunofluorescence staining was positive. Western blot and qRT-PCR results showed that the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group B were significantly higher than those in group A ( P<0.05); the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group C were significantly lower than those in group B ( P<0.05). There was no significant difference in the relative expression of HIF-1α protein and gene between groups B and D ( P>0.05); while the relative expressions of Beclin1 and LC3B proteins and genes in group D were significant lower than those in group B ( P<0.05). CONCLUSION: Hypoxia can induce the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells, and HIF-1α in rat nucleus pulposus cells under hypoxia is related to the expression of autophagy related molecules, that is, down-regulation of HIF-1α can significantly reduce the expression of autophagy related molecules, while the down-regulation of autophagy levels under hypoxia has no or little effect on the expression of HIF-1α.

Core endophyte communities of different citrus varieties from citrus growing regions in China.

The native microbiomes of citrus trees play important roles in plant health, with good communication between the native microbiome and the host plant. Here, we report on the native endophytes in 24 citrus varieties in nine citrus growing regions in China; some of the trees were healthy and others had asymptomatic or symptomatic huanglongbing, which is caused by the pathogen Candidatus Liberibacter asiaticus (CLas). We used culture-dependent analysis and characterized the isolates by partial 16S rRNA gene sequencing. The endophytes were compared between different citrus varieties, regions, and disease states (healthy, asymptomatic, and symptomatic). The total number of endophytes isolated from most of the citrus varieties was 104-106 CFU/g of leaves, but it differed significantly by disease state, with the highest numbers in the healthy leaves and the lowest in the symptomatic leaves (p < 0.05). Among the citrus varieties, the Valencia variety had the maximum number of endophyte species (22). The most dominant endophytes were Bacillus subtilis, B. velezensis, Curtobacterium luteum, and Microbacterium testaceum. The higher frequency of B. subtilis in the healthy/asymptomatic plants compared to the symptomatic plants suggests that it has a role in huanglongbing resistance. Native endophyte communities in various citrus varieties could be used to improve citrus growth and combat CLas.

Biocontrol of Soft Rot of Chinese Cabbage Using an Endophytic Bacterial Strain.

Soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) is a major constraint in the production of Chinese cabbage. The objective of this study was to demonstrate that the causative agent Pcc may be successfully managed by Bacillus amyloliquefaciens KC-1, both in vitro and in vivo. Chinese cabbage seedlings were cultivated in organic substrate termed bio-organic substrate using a floating-seedling system with B. amyloliquefaciens KC-1. This approach was applied in a greenhouse to evaluate the management of soft rot. The results showed that the extent of soft rot, as well as the transmission of Pcc to the stem progeny and its survival in the rhizosphere, was reduced following inoculation with B. amyloliquefaciens KC-1. In contrast, the population diversity of B. amyloliquefaciens KC-1 persisted in the Chinese cabbage stems after germination. These findings revealed that B. amyloliquefaciens KC-1 was able to survive and suppress the growth of Pcc in Chinese cabbage and its rhizosphere, protecting the host from the pathogen. The use of B. amyloliquefaciens KC-1 throughout the growth period of plants may be an effective strategy for the prevention of soft rot in Chinese cabbage.

Acaricidal Activity of Cyclodipeptides from Bacillus amyloliquefaciens W1 against Tetranychus urticae.

Bioassay-guided fractionation of the supernatant of the biocontrol strain Bacillus amyloliquefaciens W1 led to the isolation of eight acaricidal cyclodipeptides from the active fractions by column chromatography separation and HPLC purification. The chemical structures of these compounds were identified as cyclo-(Gly-l-Phe), 2, cyclo-(l-Phe- trans-4-OH-l-Pro), 3, cyclo-(Gly-l-Tyr), 4, cyclo-(l-Ala-l-Pro), 5, cyclo-(l-Pro- trans-4-OH-l-Pro), 6, cyclo-(Gly-l-Pro), 7, cyclo-(l-Pro-l-Pro), 8, and cyclo-(l-Tyr- trans-4-OH-l-Pro), 9. Those cyclodipeptides displayed significant acaricidal activities with LC50 values of 13.85-98.24 µM. Cyclo-(l-Tyr- trans-4-OH-l-Pro) (LC50 13.85 µM) was five times more effective than the positive control abamectin (LC50 72.06 µM). The results indicated that the hydroxyl group is an important component. This is the first report on the acaricidal capabilities of cyclodipeptides against Tetranychus urticae. The results revealed that the acaricidal activity of the biocontrol strain B. amyloliquefaciens W1 was dependent on its constituent cyclodipeptides, which have the potential to be safe and environmentally friendly acaricides.

Huanglongbing Control: Perhaps the End of the Beginning.

Huanglongbing (HLB) is one of the most destructive citrus plant diseases worldwide. It is associated with the fastidious phloem-limited α-proteobacteria 'Candidatus Liberibacter asiaticus', 'Ca. Liberibacter africanus' and 'Ca. Liberibacter americanus'. In recent years, HLB-associated Liberibacters have extended to North and South America. The causal agents of HLB have been putatively identified, and their transmission pathways and worldwide population structure have been extensively studied. However, very little is known about the epidemiologic relationships of Ca. L. asiaticus, which has limited the scope of HLB research and especially the development of control strategies. HLB-affected plants produce damaged fruits and die within several years. To control the disease, scientists have developed new compounds and screened existing compounds for their antibiotic and antimicrobial activities against the disease. These compounds, however, have very little or even no effect on the disease. The aim of the present review was to compile and compare different methods of HLB disease control with newly developed integrative strategies. In light of recent studies, we also describe how to control the vectors of this disease and the biological control of other citrus plant pathogens. This work could steer the attention of scientists towards integrative control strategies.