Bacillus paralicheniformis RP01 Enhances the Expression of Growth-Related Genes in Cotton and Promotes Plant Growth by Altering Microbiota inside and outside the Root.

Plant growth-promoting bacteria (PGPB) can promote plant growth in various ways, allowing PGPB to replace chemical fertilizers to avoid environmental pollution. PGPB is also used for bioremediation and in plant pathogen control. The isolation and evaluation of PGPB are essential not only for practical applications, but also for basic research. Currently, the known PGPB strains are limited, and their functions are not fully understood. Therefore, the growth-promoting mechanism needs to be further explored and improved. The Bacillus paralicheniformis RP01 strain with beneficial growth-promoting activity was screened from the root surface of Brassica chinensis using a phosphate-solubilizing medium. RP01 inoculation significantly increased plant root length and brassinosteroid content and upregulated the expression of growth-related genes. Simultaneously, it increased the number of beneficial bacteria that promoted plant growth and reduced the number of detrimental bacteria. The genome annotation findings also revealed that RP01 possesses a variety of growth-promoting mechanisms and a tremendous growth-promoting potential. This study isolated a highly potential PGPB and elucidated its possible direct and indirect growth-promoting mechanisms. Our study results will help enrich the PGPB library and provide a reference for plant-microbe interactions.

The Potential of Endophytes in Improving Salt-Alkali Tolerance and Salinity Resistance in Plants.

Ensuring food security for the global population is a ceaseless and critical issue. However, high-salinity and high-alkalinity levels can harm agricultural yields throughout large areas, even in largely agricultural countries, such as China. Various physical and chemical treatments have been employed in different locations to mitigate high salinity and alkalinity but their effects have been minimal. Numerous researchers have recently focused on developing effective and environmentally friendly biological treatments. Endophytes, which are naturally occurring and abundant in plants, retain many of the same characteristics of plants owing to their simultaneous evolution. Therefore, extraction of endophytes from salt-tolerant plants for managing plant growth in saline-alkali soils has become an important research topic. This extraction indicates that the soil environment can be fundamentally improved, and the signaling pathways of plants can be altered to increase their defense capacity, and can even be inherited to ensure lasting efficacy. This study discusses the direct and indirect means by which plant endophytes mitigate the effects of plant salinity stress that have been observed in recent years.

First report of Atractylodes lancea leaf spot caused by Fusarium acuminatum in China.

Atractylodes lancea Thunb. DC (cangzhu) is a traditional Chinese medicinal plant (Cai et al., 2020). In June 2020, leaf spots were observed in A. lancea plants at the Chongqing Institute of Medicinal Plant Cultivation located in Nanchuan District, Chongqing, China (29°8'26.46″ N, 107°13'23'21″ E). Approximately 75% of the plants displayed leaf spot, partial leaf wilting, and stunted growth, and some plants died. To determine the cause of this disease, five typical leaf spots were cut into small pieces. The pieces were successively surface-disinfected with 0.5% NaClO for 1 min and 75% ethanol for 30 s, washed thrice with sterile water, and placed on potato dextrose agar (PDA) to incubate at 25 ℃. These isolates initially formed abundant white aerial mycelium, then gradually developed a rose pigmentation with a brownish color in the center and grayish rose at the periphery of the colony (Li et al. 2019). Mycelial tips were picked and placed on carnation leaf agar (CLA) and inoculated for 7 days. The macroconidia of the isolates were slender, distinctively curved in the bottom half of the apical cell, and sickle-shaped, with 3-4 septa. They ranged in size from 16.68-26.49 × 1.48-2.34 µm (n=50). The microconidia were fusiform with or without one septum. Their size ranged from 6.19-11.02 × 1.25-1.43 µm (n=50) (Li et al. 2019). The morphological characteristics of the isolates were consistent with those of Fusarium spp. PCR amplification and DNA sequencing of the internal transcribed spacer (ITS) region and ß-tubulin (TUB2) gene were performed using the primers ITS1/ITS4 (White et al. 1990) and Bt-2a/Bt-2b (Robideau et al. 2011), respectively. BLASTn analysis revealed that the ITS sequences of the isolates were 100% identical to those of the F. acuminatum isolates from the Fusarium MLST database (http://isolate.fusariumdb.org/guide.php). Further analysis revealed that the TUB2 sequences were 99.14% identical to those of the F. acuminatum strain S16 isolates (MF662644) from the GeneBank database of the NCBI server. Based on the morphology and sequence analyses, the isolates were identified as F. acuminatum. Pathogenicity tests were conducted on 1.5-year-old A. lancea plants by inoculating spore suspensions under greenhouse conditions (25°C). For this, wound were made on leaves by piercing with sterilized toothpicks. 30 µl of spore suspension containing 2 × 106 conidia/ml was placed on each wound. Wounds on the leaves of control plants were inoculated with 10 µl of sterile distilled water. There were three plants for each treatment. After incubation at 25 °C for 5 days in a greenhouse, the leaves of the treated plants all showed partial wilting, consistent with the field observations. No symptoms were observed in controlled plants. The fungi were again isolated from the symptomatic tissues and were identical to the original isolate. The experiment was repeated twice with similar results. Pathogenicity symptoms were similar to what was first observed in the field and the isolated fungi were verified based on morphological characteristics, thus fulfilling Koch's postulate. To the best of our knowledge, this is the first time that A. lancea leaf spot caused by F. acuminatum has been discovered in China. The leaf spot caused by F. acuminatum on A. lancea has serious yield loss, and proper control measures should be applied.

Lysin Motif (LysM) Proteins: Interlinking Manipulation of Plant Immunity and Fungi.

The proteins with lysin motif (LysM) are carbohydrate-binding protein modules that play a critical role in the host-pathogen interactions. The plant LysM proteins mostly function as pattern recognition receptors (PRRs) that sense chitin to induce the plant's immunity. In contrast, fungal LysM blocks chitin sensing or signaling to inhibit chitin-induced host immunity. In this review, we provide historical perspectives on plant and fungal LysMs to demonstrate how these proteins are involved in the regulation of plant's immune response by microbes. Plants employ LysM proteins to recognize fungal chitins that are then degraded by plant chitinases to induce immunity. In contrast, fungal pathogens recruit LysM proteins to protect their cell wall from hydrolysis by plant chitinase to prevent activation of chitin-induced immunity. Uncovering this coevolutionary arms race in which LysM plays a pivotal role in manipulating facilitates a greater understanding of the mechanisms governing plant-fungus interactions.

Genetic dynamics of earlier maturity group emergence in south-to-north extension of Northeast China soybeans.

KEY MESSAGE: This population genetic study is characterized with direct comparisons of days to flowering QTL-allele matrices between newly evolved and originally old maturity groups of soybeans to explore its evolutionary dynamics using the RTM-GWAS procedure. The Northeast China (NEC) soybeans are the major germplasm source of modern soybean production in Americas (> 80% of the world total). NEC is a relatively new soybean area in China, expanded after its nomadic status in the seventeenth century. At nine sites of four ecoregions in NEC, 361 varieties were tested for their days to flowering (DTF), a geography-sensitive trait as an indicator for maturity groups (MGs). The DTF reduced obviously along with soybeans extended to higher latitudes, ranging in 41-83 days and MG 000-III. Using the RTM-GWAS (restricted two-stage multi-locus model genome-wide association study) procedure, 81 QTLs with 342 alleles were identified, accounting for 77.85% genetic contribution (R2 = 0.01-7.74%/locus), and other 20.75% (98.60-77.85%, h2 = 98.60%) genetic variation was due to a collective of unmapped QTLs. With soybeans northward, breeding effort made the original MG I-III evolved to MG 0-00-000. In direct comparisons of QTL-allele matrices among MGs, the genetic dynamics are identified with local exotic introduction/migration (58.48%) as the first and selection against/exclusion of positive alleles causing new recombination (40.64%) as the second, while only a few allele emergence/mutation happened (0.88%, limited in MG 0, not in MG 00-000). In new MG emergence, 24 QTLs with 19 candidate genes are the major sources. A genetic potential of further DTF shortening (13-21 days) is predicted for NEC population. The QTL detection in individual ecoregions showed various ecoregion-specific QTLs-alleles/genes after co-localization treatment (removing the random environment shifting ones).

VdNPS, a Nonribosomal Peptide Synthetase, Is Involved in Regulating Virulence in Verticillium dahliae.

Nonribosomal peptide synthetases (NPS) are known for the biosynthesis of antibiotics, toxins, and siderophore production. They are also a virulence determinant in different phytopathogens. However, until now, the functional characterization of NPS in Verticillium dahliae has not been reported. Deletion of the NPS gene in V. dahliae led to the decrease of conidia, microsclerotia, and pathogenicity. ΔVdNPS strains were tolerant to H2O2, and the genes involved in H2O2 detoxification, iron/copper transport, and cytoskeleton were differentially expressed in ΔVdNPS. Interestingly, ΔVdNPS strains exhibited hypersensitivity to salicylic acid (SA), and the genes involved in SA hydroxylation were up-regulated in ΔVdNPS compared with wild-type V. dahliae under SA stress. Additionally, during infection, ΔVdNPS induced more pathogenesis-related gene expression, higher reactive oxygen species production, and stronger SA-mediated signaling transduction in host to overcome pathogen. Uncovering the function of VdNPS in pathogenicity could provide a reliable theoretical basis for the development of cultivars with durable resistance against V. dahliae-associated diseases.

An Overview of the Molecular Genetics of Plant Resistance to the Verticillium Wilt Pathogen Verticillium dahliae.

Verticillium dahliae is a soil-borne hemibiotrophic fungus that can lead to plant vascular disease and significant economic loss worldwide. Its hosts include over 400 dicotyledon plant species, such as annual herbs, perennials, and woody plants. The average yield loss of cotton crop caused by Verticillium wilt is approximately 10-35%. As the control of this disease is an urgent task for many countries, further understanding of the interaction between plants and V. dahliae is essential. Fungi can promote or inhibit plant growth, which is important; however, the most important relationship between plants and fungi is the host-pathogen relationship. Plants can become resistant to V. dahliae through diverse mechanisms such as cell wall modifications, extracellular enzymes, pattern recognition receptors, transcription factors, and salicylic acid (SA)/jasmonic acid (JA)/ethylene (ET)-related signal transduction pathways. Over the last decade, several studies on the physiological and molecular mechanisms of plant resistance to V. dahliae have been undertaken. In this review, many resistance-related genes are summarised to provide a theoretical basis for better understanding of the molecular genetic mechanisms of plant resistance to V. dahliae. Moreover, it is intended to serve as a resource for research focused on the development of genetic resistance mechanisms to combat Verticillium wilt.

Comparative transcriptome analysis reveals regulatory networks and key genes of microsclerotia formation in the cotton vascular wilt pathogen.

Verticillium dahliae is a soil-borne, hemibiotrophic phytopathogenic fungus that causes Verticillium wilt in a broad range of economic crops. The microsclerotia (MS), which act as the main host inoculum, can survive long-term in soil resulting in uncontrollable disease. In order to clarify the mechanism of MS formation, we sequenced the whole genome-wide expression profile of V. dahliae strain V991. Compared with M1 (no MS formation), during the process of MS formation and maturation, 1354, 1571, and 1521 unique tags were significantly regulated in M2, M3, and M4 library, respectively. During MS formation, melanin synthesis-related genes were preferentially upregulated. The process is more likely to regulated by transcription factors (TFs) including C2H2, Zn2Cys6, bZIP, and fungal-specific TF domain-containing proteins; additionally, G-protein coupled receptors, Ca2+, small GTPases, and cAMP were involved in signalling transduction. Protein kinase-encoding (VDAG_06474) and synthase-encoding (VDAG_05314) genes were demonstrated to negatively and positively influence MS production, respectively. The gene expression dynamics revealed during MS formation provide comprehensive theoretical knowledge to further understanding of the metabolism and regulation of MS development in V. dahliae, potentially providing targets to control Verticillium wilt through interfering MS formation.

Characterization of VdASP F2 Secretory Factor from Verticillium dahliae by a Fast and Easy Gene Knockout System.

The vascular wilt fungus Verticillium dahliae produces persistent resting structures known as microsclerotia, which enable long-term survival of this plant pathogen in soil. The completed genome sequence of V. dahliae has facilitated large-scale investigations of individual gene functions using gene-disruption strategies based on Agrobacterium tumefaciens-mediated transformation. However, the construction of gene-deletion vectors and screening of deletion mutants have remained challenging in V. dahliae. In this study, we developed a fast and easy gene knockout system for V. dahliae using ligation-independent cloning and fluorescent screening. We identified secretory factor VdASP F2 in a T-DNA insertion library of V. dahliae and deleted the VdASP F2 gene using the developed knockout system. Phenotypic analysis suggests that VdASP F2 is not necessary for V. dahliae growth on potato dextrose agar under various stress conditions. However, on semisynthetic medium or under limited nutrient conditions at lower temperatures, the VdASP F2 deletion mutant exhibited vigorous mycelium growth, less branching, and a significant delay in melanized microsclerotial formation. Further assessment revealed that VdASP F2 was required for the expression of VDH1 and VMK1, two genes involved in microsclerotial formation. Cotton inoculated with the VdASP F2 deletion mutant wilted, demonstrating that VdASP F2 is not associated with pathogenicity under normal conditions. However, after inducing microsclerotial formation and incubation at low temperatures, cotton infected with the VdASP F2 deletion mutant did not exhibit wilt symptoms. In conclusion, our results show that VdASP F2 plays an important role in the response of V. dahliae to adverse environmental conditions and is involved in a transition to a dormant form for prolonged survival.

Purification and characterisation of an antifungal protein, MCha-Pr, from the intercellular fluid of bitter gourd (Momordica charantia) leaves.

An antifungal protein, designated MCha-Pr, was isolated from the intercellular fluid of bitter gourd (Momordica charantia) leaves during a screen for potent antimicrobial proteins from plants. The isolation procedure involved a combination of extraction, ammonium sulphate precipitation, gel filtration on Bio-Gel P-6, ion exchange chromatography on CM-Sephadex, an additional gel filtration on HiLoad 16/60 Superdex 30, and finally, HPLC on a SOURCE 5RPC column. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry indicated that the protein had a molecular mass of 25733.46Da. Automated Edman degradation was used to determine the N-terminal sequence of MCha-Pr, and the amino acid sequence was identified as V-E-Y-T-I-T-G-N-A-G-N-T-P-G-G. The MCha-Pr protein has some similarity to the pathogenesis-related proteins from Atropa belladonna (deadly nightshade), Solanum tuberosum (potato), Ricinus communis (castor bean), and Nicotiana tabacum (tobacco). Analysis of the circular dichroism spectra indicated that MCha-Pr predominantly contains α-helix and ß-sheet structures. MCha-Pr had inhibitory effects towards a variety of fungal species and the 50% inhibition of fungal growth (IC50) for Alternaria brassicae, Cercospora personata, Fusarium oxysporum, Mucor sp., and Rhizoctonia solani are 33 µM, 42 µM, 37 µM, 40 µM, and 48 µM, respectively. In addition, this antifungal protein can inhibit the germination of A. brassicae spores at 12.5 µM. These results suggest that MCha-Pr in bitter gourd leaves plays a protective role against phytopathogens and has a wide antimicrobial spectrum.

Interactions between Verticillium dahliae and its host: vegetative growth, pathogenicity, plant immunity.

Verticillium dahliae is a soil-borne phytopathogenic fungus that causes vascular wilt diseases in a wide variety of crop plants, resulting in extensive economic losses. In the past 5 years, progress has been made in elaborating the interaction between this hemibiotrophic fungus and its host plants. Some genes responsible for the vegetative growth and/or pathogenicity in V. dahliae have been identified. Plants have accrued a series of defense mechanisms, including inducible defense signaling pathways and some resistant genes to combat V. dahliae infection. Here, we have reviewed the progress in V. dahliae-plant interaction research.

Draft genome sequence of Bacillus atrophaeus TL401, a biocontrol bacterium with plant growth-promoting properties.

Bacillus atrophaeus strain TL401 exhibits biocontrol activity against Botrytis cinerea on tomato and plant growth promotion. Here, we present the draft genome sequence of strain ITL401, which includes a circular chromosome with 4,213,034 bp and a guanine-cytosine content of 43.39%.

Two modified RNA extraction methods compatible with transcript profiling and gene expression analysis for cotton roots.

Efficient isolation of high-quality RNA is of prime importance for optimal transcript profiling results and further gene expression analysis. However, it is difficult for cotton roots because of lower-than-average RNA content and high content of polysaccharides, polyphenols, and other secondary metabolites. To develop simple and reliable protocols for high-quality RNA extraction from cotton roots for transcript profiling and gene expression analysis, some modifications were introduced to a reported plant RNA isolation protocol and a reagent kit method. Using method A, we successfully extracted high-quality RNA for transcript profiling from cotton roots. Gel electrophoresis analysis and polymerase chain reaction (PCR) assay indicated that RNA had good integrity without protein and genomic DNA contamination. Furthermore, the A260/280 (1.9) and A260/230 (1.6) ratios indicated that the isolated RNA was of high purity. Using method B, about 7 µg total RNA of high quality could be obtained from 0.1 g samples from cotton roots, which can be used for reverse-transcription (RT)-PCR and quantitative real-time RT-PCR. The two RNA extraction methods were used to investigate different gene expression of cotton roots (Gossypium hirsutum) infected by weak pathogenic Verticillium dahliae and the results showed they can satisfy the transcript profiling and quantitative real-time RT-PCR requirements for RNA. Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.

Complete Genome Sequence of Bacillus licheniformis Strain GN02, Isolated from the Root Surface of Brassica chinensis.

Bacillus licheniformis GN02 was isolated from the root surface of Pak Choi Cabbage (Brassica chinensis). Here, we report the whole-genome sequence of strain GN02, which includes only a circular chromosome (4,252,022 bp; GC content, 46.08%).

Whole-Genome Sequence of the Endophytic Actinacidiphila bryophytorum Strain DS3, Isolated from the Roots of the Medicinal Plant Dysosma versipellis.

We report the whole-genome sequence of the endophytic Actinacidiphila bryophytorum strain DS3, which was isolated from the roots of the medicinal plant Dysosma versipellis. The DS3 strain genome consists of a chromosome of 8,265,668 bp, with a GC content of 72.47%, including 7,121 coding sequences.

Advances in Polysaccharide Production Based on the Co-Culture of Microbes.

Microbial polysaccharides are natural carbohydrates that can confer adhesion capacity to cells and protect them from harsh environments. Due to their various physiological activities, these macromolecules are widely used in food, medicine, environmental, cosmetic, and textile applications. Microbial co-culture is an important strategy that is used to increase the production of microbial polysaccharides or produce new polysaccharides (structural alterations). This is achieved by exploiting the symbiotic/antagonistic/chemo-sensitive interactions between microbes and stimulating the expression of relevant silent genes. In this article, we review the performance of polysaccharides produced using microbial co-culture in terms of yield, antioxidant activity, and antibacterial, antitumor, and anti-inflammatory properties, in addition to the advantages and application prospects of co-culture. Moreover, the potential for microbial polysaccharides to be used in various applications is discussed.

Pathogenesis-related protein 1 suppresses oomycete pathogen by targeting against AMPK kinase complex.

INTRODUCTION: During the arms race between plants and pathogens, pathogenesis-related proteins (PR) in host plants play a crucial role in disease resistance, especially PR1. PR1 constitute a secretory peptide family, and their role in plant defense has been widely demonstrated in both hosts and in vitro. However, the mechanisms by which they control host-pathogen interactions and the nature of their targets within the pathogen remain poorly understood. OBJECTIVES: The present study was aimed to investigate the anti-oomycete activity of secretory PR1 proteins and elaborate their underlying mechanisms. METHODS: This study was conducted in the potato-Phytophthora infestans pathosystem. After being induced by the pathogen infection, the cross-kingdom translocation of secretory PR1 was demonstrated by histochemical assays and western blot, and their targets in P. infestans were identified by yeast-two-hybrid assays, bimolecular fluorescence complementation assays, and co-immunoprecipitation assay. RESULTS: The results showed that the expression of secretory PR1-encoding genes was induced during pathogen infection, and the host could deliver PR1 into P. infestans to inhibit its vegetative growth and pathogenicity. The translocated secretory PR1 targeted the subunits of the AMPK kinase complex in P. infestans, thus affecting the AMPK-driven phosphorylation of downstream target proteins, preventing ROS homeostasis, and down-regulating the expression of RxLR effectors. CONCLUSION: The results provide novel insights into the molecular function of PR1 in protecting plants against pathogen infection, and uncover a potential target for preventing pre- and post-harvest late blight.

Identification of VdASP F2-interacting protein as a regulator of microsclerotial formation in Verticillium dahliae.

Verticillium dahliae, a notorious phytopathogenic fungus, causes vascular wilt diseases in many plant species. The melanized microsclerotia enable V. dahliae to survive for years in soil and are crucial for its disease cycle. In a previous study, we characterized the secretory protein VdASP F2 from V. dahliae and found that VdASP F2 deletion significantly affected the formation of microsclerotia under adverse environmental conditions. In this study, we clarified that VdASP F2 is localized to the cell wall. However, the underlying mechanism of VdASP F2 in microsclerotial formation remains unclear. Transmembrane ion channel protein VdTRP was identified as a candidate protein that interacts with VdASP F2 using pull-down assays followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and interaction of VdASP F2 and VdTRP was confirmed by bimolecular fluorescence complementary and coimmunoprecipitation assays. The deletion mutant was analysed to reveal that VdTRP is required for microsclerotial production, but it is not essential for stress resistance, carbon utilization and pathogenicity of V. dahliae. RNA-seq revealed some differentially expressed genes related to melanin synthesis and microsclerotial formation were significantly downregulated in the VdTRP deletion mutants. Taken together, these results indicate that VdASP F2 regulates the formation of melanized microsclerotia by interacting with VdTRP.

A Kinesin Vdkin2 Required for Vacuole Formation, Mycelium Growth, and Penetration Structure Formation of Verticillium dahliae.

The soil-borne vascular fungus Verticillium dahliae infects hundreds of dicotyledonous plants, causing severe wilt diseases. During the initial colonization, V. dahliae develops a penetration peg to enable infection of cotton roots. In some phytopathogenic fungi, vacuoles play a critical role in normal formation of the infection structure. Kinesin 2 protein is associated with vacuole formation in Ustilago maydis. To identify the function of vacuoles in the V. dahliae infection structure, we identified VdKin2, an ortholog of kinesin 2, in V. dahliae and investigated its function through gene knockout. VdKin2 mutants showed severe defects in virulence and were suppressed during initial infection and root colonization based on observation of green fluorescent protein-labeled V. dahliae. We also found that deletion of VdKin2 compromised penetration peg formation and the derived septin neck. Disruption strains were viable and showed normal microsclerotia formation, whereas mycelium growth and conidial production were reduced, with shorter and more branched hyphae. Furthermore, the VdKin2 mutant, unlike wild-type V. dahliae, lacked a large basal vacuole, accompanied by a failure to generate concentrated lipid droplets. Taken together, VdKin2 regulates vacuole formation by V. dahliae, which is required for conidiation, mycelium growth, and penetration structure formation during initial plant root infection.