First report of Cladosporium cladosporioides causing leaf blight on Sambucus chinensis in China.

Sambucus chinensis, belonging to the Caprifoliaceae family, is an economically large herb plant that is widely cultivated in southern China for its good ornamental characteristics, edible properties, and medicinal values. In July 2021, symptoms of leaf spot were observed on Sambucus chinensis plants in two fields of Chongqing Medicinal Botanical Garden (29º8'26" N, 107º13'23" E) in Nanchuan city, Chongqing, China. Disease incidence was approximately 35 and 50% for each field. The symptoms were initially yellow or black irregular spots on leaves, and then increased to larger dark brown lesions. Finally, the entire infected leaf was blighted, withering, curl and abscission. Ten blight leaves were randomly sampled from fields. Tissues were cut into small pieces and surface sterilized with 75% ethanol for 30 s and sterilized in 2% sodium hypochlorite for 2 min, rinsed thrice with sterile distilled water, plated on potato dextrose agar (PDA) plates, and incubated at 25°C for 7 days in the dark. Later, 20 isolates were obtained from the infected leaves and had similar characteristics. Three isolates were randomly selected (CQ81, CQ82, CQ83) for the further study. Colonies on PDA were olive-green to brown with a velvety texture. Conidia (n=30) were pale- to olive-brown, smooth to verruculose and produced in long, branched chains which were easily disarticulate, single celled, and elliptical to limoniform, and measured as 2.51~4.29 × 1.63~2.14 µm. Conidiophores were solitary, straight or flexous, often unbranched. The DNA of three isolates were extracted and the internal transcribed spacer (ITS) region and translation elongation factor 1-alpha (TEF1-α) were sequenced using primer pairs ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. The sequences of three isolates were 100% identical, and one representative isolate CQ82 were deposited in GenBank (ON387641, ITS; and ON409522, TEF). BLASTn analysis of these sequences showed 99 to 100% nucleotide identity with the sequences of C. cladosporioides CPC 14705 in Korea (Bensch et al. 2010). Phylogenetic analysis using Neighbor-joining method and concatenated sequences (ITS +TEF1) with MEGA7 placed isolate CQ82 in C. cladosporioides with 99% bootstrap support. On the basis of morphological and molecular characteristics, the isolates were identified as C. cladosporioides (Bensch et al. 2010; Nam et al. 2015). A total of sixteen healthy potted plants of S. chinensis were conducted for the pathogenicity test. Eight plants were selected and one shoot of each plant was randomly used for inoculation. Leaves from the shoot of each plant were brushed with 106 conidia/ml suspension of isolate CQ82. Another 8 plants were performed in the same procedure, inoculated with sterile distilled water as control. All plants were covered with plastic bags for two days and then arranged in a greenhouse with 80% relative humidity at 25°C. The pathogenicity test was repeated thrice. After 15 days inoculation, the similar symptoms were observed on the inoculated leaves, whereas controls remained healthy. The pathogen was reisolated from blight tissue and identified as C. cladosporioides by the methods described above. Although this fungus was previously reported to cause leaf disease on many plants (Meneses et al. 2018; Sun et al. 2017), this is the first report of C. cladosporioides causing leaf blight on S. chinensis in China. This study will establish a foundation for controlling the disease.

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.

First report of Phyllosticta capitalensis causing leaf spot of Mahonia fortunei in China.

Mahonia fortunei, belonging to the Berberidaceae family, is widely cultivated in fields, parks, courtyards, and roadsides for its excellent ornamental characteristics and medicinal values in southern China (Yu and Chung 2017). In May 2021, leaf spots were observed on nearly 60~80% of M. fortunei plants growing in Chongqing Normal University campus (29°36'42″N; 106°17'59″E) from Chongqing City, China. The typical symptoms on leaves were irregular spots with gray centers, brown edges, and chlorotic halos, about 1 to 7 mm in diameter, and eventually coalesced forming larger necrotic areas. Twenty symptomatic leaves were randomly sampled from five diseased plants. Tissues were cut from the lesion margins and surface sterilized in 75% ethanol for 1 min, rinsed thrice with sterile water, dried on sterilized paper, plated on potato dextrose agar (PDA) plates, and incubated at 25°C for 7 days in the dark. A total of 20 isolates were obtained from the infected leaves. Pure colonies of all fungal isolates had similar characteristics, and three isolates were randomly selected (SD11, SD18, SD19) for further study. Colonies of this fungus were olivaceous greenish to olivaceous black with a granular surface, and irregular light olive edges, finally turning black on PDA. Pycnidia were black, globose, granular, and in clusters. Conidia (n=30) were hyaline, aseptate, unicellular, obovoid to ellipsoid, narrow end with single apical appendage, and 7.5~11.2 × 4.5 ~6.5 µm. The DNA of three isolates were extracted and the internal transcribed spacer (ITS) region, actin (ACT), and translation elongation factor 1-α (TEF1) genes were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990), ACT512F/ACT783R, and ER728F/EF986R (Carbone and Kohn 1999), respectively. The sequences of three isolates were 100% identical, and one representative isolate SD18 were deposited in GenBank (ON231754, ITS; ON246259, ACT; and ON246258, TEF1). Sequence analysis revealed that the consensus sequences of ITS, ACT, and TEF1 of isolate SD18 was 99 to 100% identical to each sequence of an Indonesian strain (CBS 117118) of P. capitalensis from Musa acuminate (FJ538339 for ITS, FJ538455 for ACT, FJ538397 for TEF1). Phylogenetic analysis using Maximum Likelihood and concatenated sequences (ITS+ACT+TEF1) with MEGA7 placed isolate SD18 in P. capitalensis with 100% bootstrap support. Based on these morphological and molecular characteristics, the isolates were identified as P. capitalensis (Wikee et al. 2013). To fulfill Koch's postulates, 8 healthy potted plants were inoculated with 106 conidia/ml suspension of isolate SD18 by spraying the leaves, and another 8 plants were sprayed with sterile distilled water as control. All plants were covered with plastic bags for two days and then arranged in a greenhouse with 80% relative humidity at 25°C. The pathogenicity test was repeated thrice. After 18 days inoculation, the similar symptoms were observed on the inoculated plants, whereas control plants remained healthy. The pathogen was reisolated from symptomatic tissue and identified as P. capitalensis by the methods described above. P. capitalensis has been reported causing leaf spot on various host plants around the world (Wikee et al. 2013), recently found on tea plant, castor, and oil palm (Cheng et al. 2019; Tang et al. 2020; Nasehi et al. 2020). This is the first report of P. capitalensis causing leaf spot on M. fortune in China, and will establish a foundation for controlling the disease.

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.

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.

Psc-AFP from Psoralea corylifolia L. overexpressed in Pichia pastoris increases antimicrobial activity and enhances disease resistance of transgenic tobacco.

Psc-AFP, isolated from the seeds of Psoralea corylifolia L., is an antimicrobial protein with trypsin inhibitor activity. Its encoding gene was cloned by 3'- rapid amplification of cDNA ends (RACE) combined with Y-shaped adaptor-dependent extension (YADE) method. The gene Psc-AFP encodes a protein of 203 amino acids with a deduced signal peptide of 24 residues. The growth inhibition effect exerted by the heterologously expressed Psc-AFP in Pichia pastoris revealed that the recombinant Psc-AFP inhibited mycelium growth of Aspergillus niger, Rhizoctonia solani, and Alternaria brassicae and conidial germination of Alternaria alternata. The recombinant Psc-AFP also showed protease inhibitor activity manifested by the inhibition of trypsin. The transgenic tobacco bioassays confirmed that overexpressing Psc-AFP significantly enhanced the disease resistance of tobacco and that some of the transgenic lines were almost fully tolerant to Ralstonia solanacearum and A. alternata, whereas no apparent alteration in plant growth and development was observed. Collectively, these results indicate that the recombinant Psc-AFP is an active antimicrobial protein, with protease inhibitor activity that can be successfully produced in the yeast and tobacco and, therefore, maybe a potential antimicrobial candidate for practical use.

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.

The essential role of arginine biosynthetic genes in lunate conidia formation, conidiation, mycelial growth, and virulence of nematophagous fungus, Esteya vermicola CBS115803.

BACKGROUND: The pinewood nematode (Bursaphelenchus xylophilus) causes severe damage to pine trees. The nematophagous fungus, Esteya vermicola, exhibits considerable promise in the biological control of Bursaphelenchus xylophilus due to its infectivity. Notably, the lunate conidia produced by E. vermicola can infect Bursaphelenchus xylophilus. In the study, we aim to investigate the genes involved in the formation of the lunate conidia of E. vermicola CBS115803. RESULTS: Esteya vermicola CBS115803 yielded 95% lunate conidia on the complete medium (CM) and 86% bacilloid conidia on the minimal medium (MM). Transcriptomic analysis of conidia from both media revealed a significant enrichment of differentially expressed genes in the pathway related to 'cellular amino acid biosynthesis and metabolism'. Functional assessment showed that the knockout of two arginine biosynthesis genes (EV232 and EV289) resulted in defects in conidia germination, mycelial growth, lunate conidia formation, and virulence of E. vermicola CBS115803 in Bursaphelenchus xylophilus. Remarkably, the addition of arginine to the MM improved mycelial growth, conidiation and lunate conidia formation in the mutants and notably increased conidia yield and the lunate conidia ratio in the wild-type E. vermicola CBS115803. CONCLUSION: This investigation confirms the essential role of two arginine biosynthesis genes in lunate conidia formation in E. vermicola CBS115803. The findings also suggest that the supplementation of arginine to the culture medium can enhance the lunate conidia yield. These insights contribute significantly to the application of E. vermicola CBS115803 in managing Bursaphelenchus xylophilus infections. © 2023 Society of Chemical Industry.

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.

Establishing Gene Expression and Knockout Methods in Esteya vermicola CBS115803.

Pine wilt disease, which is caused by the nematode Bursaphelenchus xylophilus, is one of the most destructive forest diseases worldwide. Esteya vermicola, a nematophagous fungus, has emerged as a promising biological control agent. However, the limited availability of gene function analysis techniques hinders further genetic modification of this fungus. In this study, we employed a combination of enzymes (driselase, snailase, and cellulase) to enzymatically degrade the cell wall of the fungus, resulting in a high yield of protoplasts. Furthermore, by utilizing 0.6 M sucrose as an osmotic pressure stabilizer, we achieved a significant protoplast regeneration rate of approximately 31%. Subsequently, we employed the polyethylene glycol-mediated protoplast transformation method to successfully establish a genetic transformation technique for E. vermicola CBS115803. Additionally, through our investigation, we identified the Olic promoter from Aspergillus nidulans, which effectively enhanced the expression of the DsRed gene encoding a red fluorescent protein in E. vermicola CBS115803. Moreover, we successfully implemented a split-marker strategy to delete the EvIPMD gene in E. vermicola CBS115803. In summary, our findings present valuable experimental methodologies for gene function analysis in E. vermicola CBS115803.

Functional Role of RING Ubiquitin E3 Ligase VdBre1 and VdHrd1 in the Pathogenicity and Penetration Structure Formation of Verticillium dahliae.

Verticillium dahliae, a virulent soil-borne fungus, elicits Verticillium wilt in numerous dicotyledonous plants through intricate pathogenic mechanisms. Ubiquitination, an evolutionarily conserved post-translational modification, marks and labels proteins for degradation, thereby maintaining cellular homeostasis. Within the ubiquitination cascade, ubiquitin ligase E3 demonstrates a unique capability for target protein recognition, a function often implicated in phytopathogenic virulence. Our research indicates that two ubiquitin ligase E3s, VdBre1 and VdHrd1, are intrinsically associated with virulence. Our findings demonstrate that the deletion of these two genes significantly impairs the ability of V. dahliae to colonize the vascular bundles of plants and to form typical penetration pegs. Furthermore, transcriptomic analysis suggests that VdBre1 governs the lipid metabolism pathway, while VdHrd1 participates in endoplasmic-reticulum-related processes. Western blot analyses reveal a significant decrease in histone ubiquitination and histone H3K4 trimethylation levels in the ΔVdBre1 mutant. This research illuminates the function of ubiquitin ligase E3 in V. dahliae and offers fresh theoretical perspectives. Our research identifies two novel virulence-related genes and partially explicates their roles in virulence-associated structures and gene regulatory pathways. These findings augment our understanding of the molecular mechanisms inherent to V. dahliae.

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.

Correction: Yang et al. A Kinesin Vdkin2 Required for Vacuole Formation, Mycelium Growth, and Penetration Structure Formation of Verticillium dahliae. J. Fungi 2022, 8, 391.

In the original publication [...].

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.

Molecular cloning and characterization of an achene-seed-specific promoter from motherwort (Leonurus japonicus Houtt).

LJAMP1 is a small antimicrobial protein purified previously from the seeds of motherwort, and it is expressed preferentially in seeds. A 794-bp upstream sequence of the ATG start codon was isolated using a genome walking method and cloned into the upstream of the ß-glucuronidase (GUS) reporter gene to determine the GUS tissue-specific expression pattern. The transgenic tobacco showed that pLJAMP1 promoter derived GUS reporter gene special expression in pollen, achene and seed. The analysis of cis-acting elements also revealed pLJAMP1 promoter contained pollen and seed related transcriptional control elements.

Plant antimicrobial peptides: structures, functions, and applications.

Antimicrobial peptides (AMPs) are a class of short, usually positively charged polypeptides that exist in humans, animals, and plants. Considering the increasing number of drug-resistant pathogens, the antimicrobial activity of AMPs has attracted much attention. AMPs with broad-spectrum antimicrobial activity against many gram-positive bacteria, gram-negative bacteria, and fungi are an important defensive barrier against pathogens for many organisms. With continuing research, many other physiological functions of plant AMPs have been found in addition to their antimicrobial roles, such as regulating plant growth and development and treating many diseases with high efficacy. The potential applicability of plant AMPs in agricultural production, as food additives and disease treatments, has garnered much interest. This review focuses on the types of plant AMPs, their mechanisms of action, the parameters affecting the antimicrobial activities of AMPs, and their potential applications in agricultural production, the food industry, breeding industry, and medical field.

Bacillus circulans GN03 Alters the Microbiota, Promotes Cotton Seedling Growth and Disease Resistance, and Increases the Expression of Phytohormone Synthesis and Disease Resistance-Related Genes.

Plant growth-promoting bacteria (PGPB) are components of the plant rhizosphere that promote plant growth and/or inhibit pathogen activity. To explore the cotton seedlings response to Bacillus circulans GN03 with high efficiency of plant growth promotion and disease resistance, a pot experiment was carried out, in which inoculations levels of GN03 were set at 104 and 108 cfu⋅mL-1. The results showed that GN03 inoculation remarkably enhanced growth promotion as well as disease resistance of cotton seedlings. GN03 inoculation altered the microbiota in and around the plant roots, led to a significant accumulation of growth-related hormones (indole acetic acid, gibberellic acid, and brassinosteroid) and disease resistance-related hormones (salicylic acid and jasmonic acid) in cotton seedlings, as determined with ELISA, up-regulated the expression of phytohormone synthesis-related genes (EDS1, AOC1, BES1, and GA20ox), auxin transporter gene (Aux1), and disease-resistance genes (NPR1 and PR1). Comparative genomic analyses was performed between GN03 and four similar species, with regards to phenotype, biochemical characteristics, and gene function. This study provides valuable information for applying the PGPB alternative, GN03, as a plant growth and disease-resistance promoting fertilizer.