Endogenous cell wall degrading enzyme LytD is important for the biocontrol activity of Bacillus subtilis.

Autolysins are endogenous cell wall degrading enzymes (CWDEs) in bacteria that remodel the peptidoglycan layer of its own cell wall. In the Bacillus subtilis genome, at least 35 autolysin genes have been identified. However, the study of their roles in bacterial physiology has been hampered by their complexity and functional redundancy. B. subtilis GLB191 is an effective biocontrol strain against grape downy mildew disease, the biocontrol effect of which results from both direct effect against the pathogen and stimulation of the plant defense. In this study, we show that the autolysin N-acetylglucosaminidase LytD, a major autolysin of vegetative growth in B. subtilis, plays an important role in its biocontrol activity against grape downy mildew. Disruption of lytD resulted in reduced suppression of the pathogen Plasmopara viticola and stimulation of the plant defense. LytD is also shown to affect the biofilm formation and colonization of B. subtilis on grape leaves. This is the first report that demonstrates the role of an endogenous CWDE in suppressing plant disease infection of a biological control microorganism. These findings not only expand our knowledge on the biological function of autolysins but also provide a new target to promote the biocontrol activity of B. subtilis.

Genome Sequencing and Characterization of Bacillus velezensis N23 as Biocontrol Agent against Plant Pathogens.

The overuse of chemical fungicides against fungal pathogens adversely affects soil and plant health, resulting in environmental problems and food safety. Therefore, biocontrol is considered as an environmentally friendly and cost-effective green technique in environmental protection and agricultural production. We obtained a bacterial strain N23 from a contaminated plate which showed significant inhibition to anthracnose. The strain N23 was identified as Bacillus velezensis based on 16S rRNA gene, gyrA gene, and whole-genome sequence. The bacterium N23 was able to suppress the mycelial growth of numerous plant pathogenic fungi on solid media. Tomato seeds treated with strain N23 showed significantly higher germination levels than untreated ones. Moreover, strain N23 effectively reduced the lesion area of pepper anthracnose disease in planta. The gene clusters responsible for antifungal metabolites (fengycin, surfactin, and iturin) were identified in the genome sequence of N23 based on genome mining and PCR. Furthermore, methanol extracts of the bacterial culture caused significant inhibition in growth of the fungal Colletotrichum sp. and Botrytis cinerea. These findings suggested that B. velezensis N23 could be a potential biocontrol agent in agricultural production and a source of antimicrobial compounds for further exploitation.

Stable functional structure despite high taxonomic variability across fungal communities in soils of old-growth montane forests.

BACKGROUND: Major advances over the past decade in molecular ecology are providing access to soil fungal diversity in forest ecosystems worldwide, but the diverse functions and metabolic capabilities of this microbial community remain largely elusive. We conducted a field survey in montane old-growth broadleaved and conifer forests, to investigate the relationship between soil fungal diversity and functional genetic traits. To assess the extent to which variation in community composition was associated with dominant tree species (oak, spruce, and fir) and environmental variations in the old-growth forests in the Jade Dragon Snow Mountain in Yunnan Province, we applied rDNA metabarcoding. We also assessed fungal gene expression in soil using mRNA sequencing and specifically assessed the expression of genes related to organic matter decomposition and nutrient acquisition in ectomycorrhizal and saprotrophic fungi. RESULTS: Our taxonomic profiling revealed striking shifts in the composition of the saprotrophic and ectomycorrhizal guilds among the oak-, fir-, and spruce-dominated forests. The core fungal microbiome comprised only ~ 20% of the total OTUs across all soil samples, although the overlap between conifer-associated communities was substantial. In contrast, seasonality and soil layer explained only a small proportion of the variation in community structure. However, despite their highly variable taxonomic composition, fungal guilds exhibited remarkably similar functional traits for growth-related and core metabolic pathways across forest associations, suggesting ecological redundancy. However, we found that the expression profiles of genes related to polysaccharide and protein degradation and nutrient transport notably varied between and within the fungal guilds, suggesting niche adaptation. CONCLUSIONS: Overall, our metatranscriptomic analyses revealed the functional potential of soil fungal communities in montane old-growth forests, including a suite of specialized genes and taxa involved in organic matter decomposition. By linking genes to ecological traits, this study provides insights into fungal adaptation strategies to biotic and environmental factors, and sheds light on the importance of understanding functional gene expression patterns in predicting ecosystem functioning. Video Abstract.

Comprehensive genomic analysis of Bacillus paralicheniformis strain BP9, pan-genomic and genetic basis of biocontrol mechanism.

Many Bacillus species are essential antibacterial agents, but their antibiosis potential still needs to be elucidated to its full extent. Here, we isolated a soil bacterium, BP9, which has significant antibiosis activity against fungal and bacterial pathogens. BP9 improved the growth of wheat seedlings via active colonization and demonstrated effective biofilm and swarming activity. BP9 sequenced genome contains 4282 genes with a mean G-C content of 45.94% of the whole genome. A single copy concatenated 802 core genes of 28 genomes, and their calculated average nucleotide identity (ANI) discriminated the strain BP9 from Bacillus licheniformis and classified it as Bacillus paralicheniformis. Furthermore, a comparative pan-genome analysis of 40 B. paralicheniformis strains suggested that the genetic repertoire of BP9 belongs to open-type genome species. A comparative analysis of a pan-genome dataset using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Cluster of Orthologous Gene groups (COG) revealed the diversity of secondary metabolic pathways, where BP9 distinguishes itself by exhibiting a greater prevalence of loci associated with the metabolism and transportation of organic and inorganic substances, carbohydrate and amino acid for effective inhabitation in diverse environments. The primary secondary metabolites and their genes involved in synthesizing bacillibactin, fencing, bacitracin, and lantibiotics were identified as acquired through a recent Horizontal gene transfer (HGT) event, which contributes to a significant part of the strain`s antimicrobial potential. Finally, we report some genes essential for plant-host interaction identified in BP9, which reduce spore germination and virulence of multiple fungal and bacterial species. The effective colonization, diverse predicted metabolic pathways and secondary metabolites (antibiotics) suggest testing the suitability of strain BP9 as a potential bio-preparation in agricultural fields.

Effects of rice blast biocontrol strain Pseudomonas alcaliphila Ej2 on the endophytic microbiome and proteome of rice under salt stress.

Introduction: Soil salinity is a prevalent environmental stress in agricultural production. Microbial inoculants could effectively help plants to alleviate salt stress. However, there is little knowledge of the biocontrol strain Pseudomonas alcaliphila Ej2 mechanisms aiding rice plants to reduce the adverse effects caused by salt stress. Methods: We performed integrated field and greenhouse experiments, microbial community profiling, and rice proteomic analysis to systematically investigate the Ej2 mechanism of action. Results: The results displayed that biocontrol strain Ej2 increased shoot/root length and fresh/dry weight compared with control under salt stress. Meanwhile, strain Ej2 has the ability to control rice blast disease and promote rice growth. Furthermore, the microbial community analysis revealed that the alpha-diversity of Ej2-inoculated plants was higher than the control plants, expect the Shannon index of the bacterial microbiome and the Ej2-inoculated samples clustered and separated from the control samples based on beta-diversity analysis. Importantly, the enriched and specific OTUs after Ej2 inoculation at the genus level were Streptomyces, Pseudomonas, Flavobacterium, and Bacillus. Moreover, we observed that Ej2 inoculation influenced the rice proteomic profile, including metabolism, plant-pathogen interactions, and biosynthesis of unsaturated fatty acids. These results provide comprehensive evidence that Ej2 inoculation induced the rice endophytic microbiome and proteomic profiles to promote plant growth under salt stress. Discussion: Understanding the biocontrol strain effects on the endophytic microbiome and rice proteomics will help us better understand the complex interactions between plants and microorganisms under salt stress. Furthermore, unraveling the mechanisms underlying salt tolerance will help us more efficiently ameliorate saline soils.

The bacterial and fungal microbiomes of ectomycorrhizal roots from stone oaks and Yunnan pines in the subtropical forests of the Ailao Mountains of Yunnan.

Ectomycorrhizal (ECM) symbioses play an important role in tree biology and forest ecology. However, little is known on the composition of bacterial and fungal communities associated to ECM roots. In the present study, we surveyed the bacterial and fungal microbiome of ECM roots from stone oaks (Lithocarpus spp.) and Yunnan pines (Pinus yunnanensis) in the subtropical forests of the Ailao Mountains (Yunnan, China). The bacterial community was dominated by species pertaining to Rhizobiales and Acidobacteriales, whereas the fungal community was mainly composed of species belonging to the Russulales and Thelephorales. While the bacterial microbiome hosted by ECM roots from stone oaks and Yunnan pines was very similar, the mycobiome of these host trees was strikingly distinct. The microbial networks for bacterial and fungal communities showed a higher complexity in Lithocarpus ECM roots compared to Pinus ECM roots, but their modularity was higher in Pinus ECM roots. Seasonality also significantly influenced the fungal diversity and their co-occurrence network complexity. Our findings thus suggest that the community structure of fungi establishing and colonizing ECM roots can be influenced by the local soil/host tree environment and seasonality. These results expand our knowledge of the ECM root microbiome and its diversity in subtropical forest ecosystems.

Pseudomonas spp. Enriched in Endophytic Community of Healthy Cotton Plants Inhibit Cotton Verticillium Wilt.

The plant microbiome plays a fundamental role in plant growth and health. However, detailed information regarding the plant endophytic microbiome during the infection period of a pathogen is largely unknown. Here, we investigated the microbial community of healthy and diseased cotton plants and the root exudate profiles of susceptible and resistant cultivars utilizing high-throughput sequencing and metabolomics. The results showed that the pathogen infection reduced bacterial diversity and significantly affected the bacterial community composition. The microbiome assembly is shaped predominantly by cultivars. The endophytic microbiome of the infected plants showed greater complexity than the healthy plants in network analysis. The results displayed that a total of 76 compounds were significantly different in the two groups, with 18 compounds showing a higher relative abundance in the resistant cultivars and 58 compounds in the susceptible cultivars. Pathway enrichment analysis showed that pathways related to plant hormone signal transduction, biosynthesis of various secondary metabolites, and biosynthesis and metabolism of amino acids were prominently altered. We also demonstrate that plants inoculated with Pseudomonas sp. strains showed increased resistance to the cotton Verticillium wilt compared with the control plants in pot experiments. Overall, it showed that the pathogen infection affected the community composition, and healthy plants displayed an enriched beneficial microbiome to combat the plant disease. These findings significantly advance our understanding of the endophytic microbiome assembly under the pathogen infection and develop microbiome-based solutions for sustainable crop production systems.

Ecological Drivers of the Soil Microbial Diversity and Composition in Primary Old-Growth Forest and Secondary Woodland in a Subtropical Evergreen Broad-Leaved Forest Biome in the Ailao Mountains, China.

Replacement of primary old-growth forests by secondary woodlands in threatened subtropical biomes drives important changes at the level of the overstory, understory and forest floor, but the impact on belowground microbial biodiversity is yet poorly documented. In the present study, we surveyed by metabarcoding sequencing, the diversity and composition of soil bacteria and fungi in the old-growth forest, dominated by stone oaks (Lithocarpus spp.) and in the secondary Yunnan pine woodland of an iconic site for biodiversity research, the Ailaoshan National Nature Reserve (Ailao Mountains, Yunnan province, China). We assessed the effect of forest replacement and other environmental factors, including soil horizons, soil physicochemical characteristics and seasonality (monsoon vs. dry seasons). We showed that tree composition and variation in soil properties were major drivers for both bacterial and fungal communities, with a significant influence from seasonality. Ectomycorrhizal Operational Taxonomic Units (OTUs) dominated the functional fungal guilds. Species richness and diversity of the bacterial and fungal communities were higher in the pine woodland compared to the primary Lithocarpus forest, although prominent OTUs were different. The slightly lower complexity of the microbiome in the primary forest stands likely resulted from environmental filtering under relatively stable conditions over centuries, when compared to the secondary pine woodlands. In the old-growth forest, we found a higher number of species, but that communities were homogeneously distributed, whereas in the pine woodlands, there is a slightly lower number of species present but the communities are heterogeneously distributed. The present surveys of the bacterial and fungal diversity will serve as references in future studies aiming to assess the impact of the climate change on soil microbial diversity in both old-growth forests and secondary woodlands in Ailaoshan.

Comprehensive Genomic Analysis of the Endophytic Bacillus altitudinis Strain GLB197, a Potential Biocontrol Agent of Grape Downy Mildew.

Bacillus has been extensively studied for agricultural application as a biocontrol agent. B. altitudinis GLB197, an endophytic bacterium isolated from grape leaves, exhibits distinctive inhibition to grape downy mildew based on unknown mechanisms. To determine the genetic traits involved in the mechanism of biocontrol and host-interaction traits, the genome sequence of GLB197 was obtained and further analyzed. The genome of B. altitudinis GLB197 consisted of one plasmid and a 3,733,835-bp circular chromosome with 41.56% G + C content, containing 3,770 protein-coding genes. Phylogenetic analysis of 17 Bacillus strains using the concatenated 1,226 single-copy core genes divided into different clusters was conducted. In addition, average nucleotide identity (ANI) values indicate that the current taxonomy of some B. pumilus group strains is incorrect. Comparative analysis of B. altitudinis GLB197 proteins with other B. altitudinis strains identified 3,157 core genes. Furthermore, we found that the pan-genome of B. altitudinis is open. The genome of B. altitudinis GLB197 contains one nonribosomal peptide synthetase (NRPS) gene cluster which was annotated as lichenysin. Interestingly, the cluster in B. altitudinis has two more genes than other Bacillus strains (lgrD and lgrB). The two genes were probably obtained via horizontal gene transfer (HGT) during the evolutionary process from Brevibacillus. Taken together, these observations enable the future application of B. altitudinis GLB197 as a biocontrol agent for control of grape downy mildew and promote our understanding of the beneficial interactions between B. altitudinis GLB197 and plants.

Comprehensive genomic analysis of Bacillus velezensis AL7 reveals its biocontrol potential against Verticillium wilt of cotton.

Verticilllium wilt of cotton is a devastating soil-borne disease, which is caused by Verticillium dahliae Kleb. Bacillus velezensis strain AL7 was isolated from cotton soil. This strain efficiently inhibited the growth of V. dahliae. But the mechanism of the biocontrol strain AL7 remains poorly understood. To understand the possible genetic determinants for biocontrol traits of this strain, we conducted phenotypic, phylogenetic and comparative genomics analysis. Phenotypic analysis showed that strain AL7 exhibited broad-spectrum antifungal activities. We determined that the whole genome sequence of B. velezensis AL7 is a single circular chromosome that is 3.89 Mb in size. The distribution of putative gene clusters that could benefit to biocontrol activities was found in the genome. Phylogenetic analysis of Bacillus strains by using single core-genome clearly placed strain AL7 into the B. velezensis. Meantime, we performed comparative analyses on four Bacillus strains and observed subtle differences in their genome sequences. In addition, comparative genomics analysis showed that the core genomes of B. velezensis are more abundant in genes relevant to secondary metabolism compared with B. subtilis strains. Single mutant in the biosynthetic genes of fengycin demonstrated the function of fengycin in the antagonistic activity of B. velezensis AL7. Here, we report a new biocontrol bacterium B. velezensis AL7 and fengycin contribute to the biocontrol efficacy of the strain. The results showed in the research further sustain the potential of B. velezensis AL7 for application in agriculture production and may be a worthy biocontrol strain for further studies.

Synthetic community with six Pseudomonas strains screened from garlic rhizosphere microbiome promotes plant growth.

The rhizosphere microbiome plays an important role in the growth and health of many plants, particularly for plant growth-promoting rhizobacteria (PGPR). Although the use of PGPR could improve plant production, real-world applications are still held back by low-efficiency methods of finding and using PGPR. In this study, the structure of bacterial and fungal rhizosphere communities of Jinxiang garlic under different growth periods (resume growth, bolting and maturation), soil types (loam, sandy loam and sandy soil) and agricultural practices (with and without microbial products) were explored by using amplicon sequencing. High-efficiency top-down approaches based on high-throughput technology and synthetic community (SynCom) approaches were used to find PGPR in garlic rhizosphere and improve plant production. Our findings indicated that Pseudomonas was a key PGPR in the rhizosphere of garlic. Furthermore, SynCom with six Pseudomonas strains isolated from the garlic rhizosphere were constructed, which showed that they have the ability to promote plant growth.

Screening and Application of Bacillus Strains Isolated from Nonrhizospheric Rice Soil for the Biocontrol of Rice Blast.

Rice blast, caused by Magnaporthe oryzae, is one of the most destructive rice diseases worldwide. The aim of this study was to screen bacterial isolates to efficiently prevent the occurrence of rice blast. A total of 232 bacterial isolates were extracted from nonrhizospheric rice soil and were screened for antifungal activity against M. oryzae using a leaf segment assay. Strains S170 and S9 showed significant antagonistic activity against M. oryzae in vitro and in leaf disk assays, and controlled M. oryzae infection under greenhouse conditions. The results showed that strains S170 and S9 could effectively control rice leaf blast and panicle neck blast after five spray treatments in field. This suggested that the bacterial strains S170 and S9 were valuable and promising for the biocontrol of rice disease caused by M. oryzae. Based on 16S rDNA, and gyrA and gyrB gene sequence analyses, S170 and S9 were identified as Bacillus amyloliquefaciens and B. pumilus, respectively. The research also demonstrated that B. amyloliquefaciens S170 and B. pumilus S9 could colonize rice plants to prevent pathogenic infection and evidently suppressed plant disease caused by 11 other plant pathogenic fungi. This is the first study to demonstrate that B. amyloliquefaciens and B. pumilus isolated from nonrhizospheric rice soil are capable of recolonizing internal rice stem tissues.

Complete genome sequence data of Bacillus pumilus GLB197, an effective antagonist of grape downy mildew.

Bacillus pumilus GLB197 isolated from grape leaves exhibited strong inhibitory activity against grape downy mildew. The whole genome of the strain was sequenced to gain new insights into its molecular mechanism underlying the biocontrol on phytopathogens. The complete genome contains one chromosome (3,733,835 bp) and one plasmid (7061 bp). Several gene clusters related to biosynthesis of antimicrobial compounds were predicted. The genome provides insights into the possible biocontrol mechanisms and further application of this specific bacterium.

Draft genome sequence data of Bacillus subtilis strain 9407, isolated from healthy apples in China.

Bacillus subtilis strain 9407 is an endophyte which was isolated from healthy apples from an infested orchard that exhibits strong inhibitory activity against Botryosphaeria dothidea. Whole-genome sequencing of B. subtilis 9407 was performed using the Illumina Hiseq platforms. Here, we report the draft genome sequence of B. subtilis strain 9407 containing 16 scaffolds (4,062,615 bp), 4033 coding sequences, and an average 43.66% G + C content. The genome contains genes responsible for the production of several bioactive secondary metabolites, including the lipopeptides fengycin and surfactin. The genome information will provide fundamental knowledge of the organism. This whole-genome shotgun data has been deposited at DDBJ/EMBL/GenBank under the accession numbers PISO00000000.

Complete Genome Sequence of Bacillus velezensis Strain AL7, a Biocontrol Agent for Suppression of Cotton Verticillium Wilt.

Bacillus velezensis AL7, isolated from cotton soil, had strong antagonistic activity to Verticillium dahlia Kleb. The AL7 genome consisted of one chromosome with 3,894,709 bp (46.64% G+C content). Genome annotation predicted 3,706 protein-coding genes, 86 tRNAs, and 27 rRNAs. We sequenced and annotated the complete AL7 genome to help us better understand use of this strain.

Draft Genome Sequence of an Endophytic Biocontrol Bacterium, Bacillus velezensis PG12, Isolated from Apple Fruit.

Bacillus velezensis PG12 is a biocontrol endophytic bacterium that is capable of inhibition of a broad range of plant-pathogenic fungi. The strain has the potential to be developed into a biocontrol agent for use in agriculture. Here, we report the draft genome sequence of B. velezensis PG12, which contains 22 scaffolds (3,990,845 bp), 3,884 coding sequences (CDSs), and an average G+C content of 46.45%.

Intraocular involvement is associated with a high risk of disease relapse in primary central nervous system lymphoma.

The aim of the present study was to prospectively evaluate the recurrence and survival outcome of primary central nervous system lymphoma (PCNSL) with intraocular involvement. For this purpose, a prospective cohort of 103 pathologically confirmed patients with PCNSL was enrolled in this study. Ophthalmologic examinations were performed both at diagnosis and during follow­up. The patients with PCNSL with suspected intraocular involvement underwent vitrectomy for confirmation. Patients who presented with intraocular involvement either at diagnosis or during disease progression were allocated to the intraocular lymphoma (IOL) group. All patients with PCNSL received systemic methotrexate (MTX)­based chemotherapy with or without radiotherapy. MTX intravitreal injection combined with systemic MTX­based chemotherapy was recommended once ocular lesions were confirmed. Recurrent intraocular and central nervous system (CNS) events, progression­free survival (PFS) and overall survival (OS) outcomes were analyzed. The findings of this study revealed that 21 patients with PCNSL exhibited intraocular involvement. One patient with IOL presented with isolated ocular lymphoma at the initial diagnosis, and the others presented with ocular involvement along with CNS invasion during the course of the disease. A total of 14 patients received systemic MTX­based chemotherapy prior to the diagnosis of IOL. The recurrence rates in patients with or without intraocular involvement were 71.4 and 46.3%, respectively (P=0.04) and the relapse sites in the patients with IOL included the brain (3 patients), eyes (6 patients), and both brain and eyes (6 cases). The median PFS was 13 months in the IOL group and 19 months in the patients without intraocular involvement (non­IOL) (P=0.019). The median OS was 51 months vs. 56 months, respectively (P=0.312). There was no significant difference in the 2­year PFS and OS rates between the 2 groups (23.8% vs. 23.2%, P=0.951; and 61.9% vs. 41.4%, P=0.093, respectively). On the whole, the findings of this study suggest that patients with IOL have a high risk of relapse and a poor PFS compared to patients without IOL, but a similar OS.

Comparative genomic and functional analyses of four sequenced Bacillus cereus genomes reveal conservation of genes relevant to plant-growth-promoting traits.

Some Bacillus strains function as predominant plant-growth-promoting rhizobacteria. Bacillus cereus 905 is a rod-shaped Gram-positive bacterium isolated from wheat rhizosphere and is a rhizobacterium that exhibits significant plant-growth-promoting effects. Species belonging to the genus Bacillus are observed in numerous different habitats. Several papers on B. cereus are related to pathogens that causes food-borne illness and industrial applications. However, genomic analysis of plant-associated B. cereus has yet to be reported. Here, we conducted a genomic analysis comparing strain 905 with three other B. cereus strains and investigate the genomic characteristics and evolution traits of the species in different niches. The genome sizes of four B. cereus strains range from 5.38 M to 6.40 M, and the number of protein-coding genes varies in the four strains. Comparisons of the four B. cereus strains reveal 3,998 core genes. The function of strain-specific genes are related to carbohydrate, amino acid and coenzyme metabolism and transcription. Analysis of single nucleotide polymorphisms (SNPs) indicates local diversification of the four strains. SNPs are unevenly distributed throughout the four genomes, and function interpretation of regions with high SNP density coincides with the function of strain-specific genes. Detailed analysis indicates that certain SNPs contribute to the formation of strain-specific genes. By contrast, genes related to plant-growth-promoting traits are highly conserved. This study shows the genomic differences between four strains from different niches and provides an in-depth understanding of the genome architecture of these species, thus facilitating genetic engineering and agricultural applications in the future.

Spontaneous remission in patients with acute myeloid leukemia with t(8;21) or cutaneous myeloid sarcoma: two case reports and a review of the literature.

Spontaneous remission (SR) in patients with acute myeloid leukemia (AML) is rare. We herein present two such cases. The first case was of AML-M2 accompanied by a bone marrow cytogenetic analysis revealing 46, XY, t(8;21)(q22,q22). The second case was of isolated cutaneous myeloid sarcoma (MS) that progressed to AML within seven months. Both of the patients had symptoms of infection and anemia and were therefore treated with antibiotics and transfusions. The SR lasted for two months and one month, respectively. Currently, the mechanisms underlying SR remain ambiguous. Possible underlying mechanisms with a review of the related literature are discussed.