Vascular Wilt Disease Research Articles

Comparative proteomic analysis identifies potential pathogenicity/virulence factors in Fusarium oxysporum f.sp. ricini inciting vascular wilt disease in castor

Fusarium oxysporum f.sp. ricini (FOR), the causal agent of castor wilt, is the major pathogen of castor. However, there is meagre information regarding FOR pathogenesis and virulence. Development of effective disease management strategies requires a deep understanding of the pathogenicity and virulence factors deployed by the pathogen to infect its host. We have compared the mycelial proteomes of FOR isolates showing differential virulence, to identify potential pathogenicity and virulence factors. Differentially accumulated/upregulated protein spots in the virulent isolate were further identified by MALDI-TOF/TOF. The identified proteins showed homology with the fungal proteins that play crucial roles in pathogen penetration, colonization, invasion, degradation of plant cutin, detoxification of host-generated ROS, activation of signal transduction pathways, stress response, post-translational modification and toxin production. Our study suggested that the identified proteins might contribute to the high degree of virulence of the virulent FOR isolates.

Spectral characterization and classification of two different crown root rot and vascular wilt diseases (fusarium oxysporum f.sp. radicis lycopersici and fusarium solani) in tomato plants using different machine learning algorithms

Spectral characterization and classification of two different crown root rot and vascular wilt diseases (fusarium oxysporum f.sp. radicis lycopersici and fusarium solani) in tomato plants using different machine learning algorithms

Two Metalloproteases VdM35-1 and VdASPF2 from Verticillium dahliae Are Required for Fungal Pathogenicity, Stress Adaptation, and Activating Immune Response of Host.

Verticillium dahliae is a soilborne fungus that causes destructive vascular wilt diseases in a wide range of plant hosts. In this study, we identified two M35 family metalloproteinases: VdM35-1 and VdASPF2, and investigated their function in vitro and in vivo. The results showed that VdM35-1 and VdASPF2 were located in the cell membrane, as secreted proteins depended on signal peptide, and two histidine residues (H) induced cell death and activated plant immune response. VdM35-1 depended on membrane receptor proteins NbBAK1 and NbSOBIR1 in the process of inducing cell death, while VdASPF2 did not depend on them. The deletion of VdM35-1 and VdASPF2 led to the decrease of sporulation and the slow shortening of mycelial branch growth, and the spore morphology of VdM35-1-deficient strain became malformed. In addition, ΔVdM35-1 and ΔVdASPF2 showed more sensitive to osmotic stress, SDS, Congo red (CR), and high temperature. In terms of the utilization of carbon sources, the knockout mutants exhibited decreased utilization of carbon sources, and the growth rates on the medium containing sucrose, starch, and pectin were lower than the wild type strain, with significantly limited growth, especially on galactose-containing medium. Furthermore, ΔVdM35-1 and ΔVdASPF2 showed a significant reduction in pathogenicity. Collectively, these results suggested that VdM35-1 and VdASPF2 were important multifunction factors in the pathogenicity of V. dahliae and relative to stress adaptation and activated plant immune response. IMPORTANCE Verticillium wilt, caused by the notorious fungal pathogen V. dahliae, is one of the main limiting factors for agricultural production. Metalloproteases played an important role in the pathogenic mechanism of pathogens. Our research found that M35 family metalloproteases VdM35-1 and VdASPF2 played an important role in the development, adaptability, and pathogenicity of V. dahliae, providing a new perspective for further understanding the molecular mechanism of virulence of fungal pathogens.

Reaction of inbred lines of habanero hot pepper (Capsicum chinense Jacq.) to inoculation of Fusarium oxysporum

Objective: To evaluate the reaction of five inbred lines of habanero pepper (Capsicum chinense Jacq.) to the inoculation of Fusarium oxisporum and to identify genotypes with possible levels of resistance to this pathogen. Design/methodology/approach: Samples of hot pepper plant stems with typical symptoms of vascular wilt disease were obtained. Then, due to microscopic morphological characteristics of mycelium and conidia in the samples, patogen was identified as Fusarium oxisporum. By immersion of roots, at a concentration of 1x106 conidia per mL, five inbred lines of habanero pepper were inoculated. In addition to the serrano type cultivar Criollo de Morelos 334 (CM-334). The percentage disease index and area under the disease progress curve (ABCPE) were estimated in a randomized complete block design, with three replicates. Results: Significant differences (p≤0.05) were observed for disease index and ABCPE in the genotypes evaluated; evidencing the difference in the genetic basis of the genotypes and the resistance or susceptibility to Fusarium oxisporum. The CM-334 cultivar, the Habanero 5 and Habanero 8 lines showed the lowest percentage of incidence of the disease (10, 20 and 30%, respectively) and ABCPE of 300, 560 and 880. In turn, lines Habanero 9 and Habanero 6 reached the highest ABCPE values with 2220 and 2190, respectively. Study limitations/implications: The disease resistance response in plants is complex and contains multiple interactions between genes, proteins, and metabolites. Findings/conclusions: The grouping of genotypes according to their reaction to Fusarium wilt disease facilitated the identification of resistant and susceptible lines, which can be considered in subsequent genetic improvement studies for hot pepper cultivation.

Response of seed endophytic bacteria for the management of chickpea wilt, Fusarium oxysporum f. sp. ciceris

Vascular wilts are devastating plant diseases that can affect both annual crops as well as woody perennials, hence inducing major food losses and damaging valuable natural ecosystems. Because of ecological and economic reasons, the management of vascular wilt diseases by conventional chemical methods is raising concerns. More environmentally friendly alternatives such as the use of microbial antagonist to control phytopathogens are now of growing interest. The fact that bacterial endophytes are able to colonize an ecological niche similar to that of vascular wilt pathogens favours them as potential biocontrol agents against wilt diseases. Several possible disease suppression mechanisms of beneficial bacteria were proposed, among them induction of systemic resistance, growth promotion and competition. In this view, we studied out the seed endophytic bacteria (SEB) for the management of chickpea wilt Caused by Fusarium oxysporum f. sp. ciceris. Among the treatment of seed entophytic bacterial isolate in SEB-5 was found highest germination percent (92% in JG 11), minimum mortality percent at pre and post–emergence (7.69 and 9.53% in JG 11 and JG 16, respectively). The minimum total mortality percent 17.82 % was found in JG 11 of SEB-5. The other seed entophytic bacterial isolate were found statistically at par with SEB-5. The minimum germination percent (78.5% in JG 62) as well as highest total mortality percent approximate 58% in JG 11 was found in T6 (treated control). Similarly, all the treatments of seed entophytic bacterial had higher root length and shoot length as compared to T6. Among the treatments the SEB-5 was found significantly highest by 23 cm in root length and 48 cm in shoot length as well as highest vigour index (6613) over the T6 in JG 11 variety. Among the varieties grown the JG 11 was perfumed better and found resistant and JG 62 was found susceptible to wilt. The treatment of seed endophytic bacterial isolate SEB-5, SEB-3, SEB-2, SEB-1 and SEB-4 was identified against wilt pathogen in reduce disease incidence, plant mortality and severity as well as promoting plant growth and health.

Aerial Branch Sampling to Detect Forest Pathogens

Diagnostic testing to detect forest pathogens requires the collection of physical samples from affected trees, which can be challenging in remote or rugged environments. As an alternative to traditional ground-based sampling at breast height by field crews, we examined the feasibility of aerially sampling and testing material collected from upper canopy branches using a small unoccupied aerial system (sUAS). The pathogen of interest in this study is Ceratocystis lukuohia, the fungal pathogen responsible for Ceratocystis wilt of ‘ōhi‘a, a vascular wilt disease which has caused widespread mortality to ‘ōhi‘a in native forests across the state of Hawai‘i. To characterize the minimum branch diameter needed to successfully detect the pathogen of interest in infected trees, we tested 63 branch samples (0.8–9.6 cm in diameter) collected from felled trees inoculated with C.lukuohia on Hawai‘i Island. Subsequently, we aerially sampled branches from ten symptomatic ‘ōhi‘a (Metrosideros polymorpha) trees using two different branch sampling systems, the Flying Tree Top Sampler from ETH Zurich and the new Kūkūau branch sampler system introduced in this work, producing 29 branch samples with a maximum diameter of 4.2 cm and length of >2 m. We successfully detected the target fungal pathogen from the collected branches and found that branch diameter, leaf presence and condition, as well as wood moisture content are important factors in pathogen detection in sampled branches. None of the smallest branch samples (those <1 cm in diameter) tested positive for C.lukuohia, while 77% of the largest diameter branch samples (5–10 cm) produced positive results. The Kūkūau branch sampler system is capable of retrieving branches up to 7 cm diameter, providing important capacity for pathogenic research requiring larger diameter samples for successful diagnostic testing. Inconclusive and/or non-detection laboratory results were obtained from sample materials that were either too desiccated or from a branch with asymptomatic leaves, suggesting there is an optimal temporal window for sampling.

Markerless gene deletion in Ralstonia solanacearum based on its natural transformation competence.

Ralstonia solanacearum species complex (RSSC) is a group of Gram-negative bacterial pathogen capable of infecting numerous plants and crops, causing severe vascular wilt diseases. Functional analysis of the genes associated with bacterial virulence is critical for elucidating the molecular mechanisms that govern the bacterial pathogenicity. To this end, an efficient gene deletion method would be of great help. In this study, we set to develop an efficient and simple markerless gene deletion method by exploiting its natural transformation competence and the FLP/FRT recombination system. We found that natural transformation using PCR products provided much higher transformation frequency than the plasmid-based triparental mating and electroporation. We thus generated the gene deletion fusion PCR fragments by incorporating the upstream and downstream DNA fragments of the target gene and an antibiotic resistance gene flanked by FRT sites, and delivered the PCR products into R. solanacearum cells through natural transformation. Using this method, we knocked out the epsB and phcA genes, which are associated with exopolysaccharide (EPS) biosynthesis and regulation, respectively, in several R. solanacearum strains isolated from different host plants at a frequency from 5 (1E-08) to 45 (1E-08). To remove the antibiotic marker gene, the plasmid expressing the FLP enzyme was introduced into the above knockout mutants, which enabled removal of the marker gene. The effective combination of natural transformation and the FLP/FRT recombination system thus offers a simple and efficient method for functional study of putative virulence genes and for elucidation of R. solanacearum pathogenic mechanisms.

Scarcity of major resistance genes against Verticillium wilt caused by Verticillium dahliae

AbstractVerticillium dahliae is a soil‐borne fungal pathogen that causes vascular wilt disease in numerous plant species. The only described qualitative resistances against V. dahliae are the Ve1 gene and the V2 locus in tomato. These resistances have been overcome by virulent strains. We tried to identify additional resistances. Out of the methods we tested, comparing the canopy area of V. dahliae‐inoculated plants with mock‐inoculated plants yielded the best discriminative power in resistance tests. Out of six wild tomato accessions that were previously reported to possess some resistance, Solanum pimpinellifolium G1.1596 and Solanum cheesmanii G1.1615 displayed the lowest stunting and the least colonization by V. dahliae. Recombinant inbred line (RIL) populations were developed of both populations. No QTLs were identified in the G1.1596 RIL population. In the G1.1615 population, four small‐effect QTLs were associated with reduced stunting. Many studies in other hosts also failed to discover major resistance genes against V. dahliae. We hypothesize that the scarcity of major resistance genes against V. dahliae is caused by its endophytic behaviour in nature. The limited damage in nature would not lead to evolutionary pressure to evolve major resistances. However, in agriculture, V. dahliae can behave more pathogenic, leading to serious damage.

In Search of Antifungals from the Plant World: The Potential of Saponins and Brassica Species against Verticillium dahliae Kleb.

Control methods alternative to synthetic pesticides are among the priorities for both organic and conventional farming systems. Plants are potential sources of compounds with antimicrobial properties. In this study, the antifungal potentialities of saponins derived from Medicago species and oat grains and of brassica sprouts have been explored for the control of Verticillium dahliae, a widely distributed fungal pathogen that causes vascular wilt disease on over 200 plant species. All the tested plant extracts showed antifungal properties. Such compounds, able to reduce mycelium growth and conidia formation, deserve deeper in vivo evaluation, even in combination with a delivery system.

DEVELOPMENT OF SPECIFIC PRIMERS FOR 16S rRNA GENE ANALYSIS IN THE DETECTION OF Ralstonia solanacearum SPECIES COMPLEX

Members of Ralstonia solanacearum species complex (RSSC) are causal agents of vascular wilt disease in more than 450 crop species, including solanaceous plants such as potatoes, tomatoes, bell pepper, eggplant, etc. These phytopathogens cause serious yield loss mostly in solanaceous crops which are grown in tropical, subtropical, and temperate regions of the world. Yield losses comprise 80%–100% in potato, up to 91% for tomato, 10%–30% in tobacco, 33%–90% in banana, and reduce crop productivity and yield. PCR-methods are specific, sensitive and cost-effective approaches for the detection and identification of RSSC members. The objective of this study was to compare specificity of routinely used primer mix for PCR RSSC detection with the newly developed pairs of species-specific primers for ease of use diagnostics in a laboratory. Materials and Methods. The conserved genomic regions of the 16S rRNA sequences of R. solanacearum, R. pseudosolanacearum, and R. syzygii were selected for the design of primers for this study. Newly created primer species specificity was tested in PCR using the DNA of the two targets and 13 non-target strains of bacteria. Results. Three pairs of newly created primers Rs-28(F)/Rs-193(R), Rs-28(F)/OLI-160(R), Rs28(F)/OLI248(R) produced single specific fragments for bacterial strains of Ralstonia solanacearum: 166 bp, 132 bp, and 220 bp. products respectively. No PCR products were obtained during amplification with the negative control or non-target DNA templates from other bacterial species. Conclusion. Designed primers can be used for the development of PCR system for the qualitative and quantitative detection of RSSC members.

Comparative transcriptome analysis reveals the resistance regulation mechanism and fungicidal activity of the fungicide phenamacril in Fusarium oxysporum

Fusarium oxysporum (Fo) is an important species complex of soil-borne pathogenic fungi that cause vascular wilt diseases of agricultural crops and some opportunistic diseases of humans. The fungicide phenamacril has been extensively reported to have antifungal activity against Fusarium graminearum and Fusarium fujikuroi. In this study, we found that the amino acid substitutions (V151A and S418T) in Type I myosin FoMyo5 cause natural low resistance to phenamacril in the plant pathogenic Fo isolates. Therefore, we compared the transcriptomes of two phenamacril-resistant Fo isolates FoII5, Fo1st and one phenamacril-sensitive isolate Fo3_a after 1 μg/mL phenamacril treatment. Among the 2728 differentially expressed genes (DEGs), 14 DEGs involved in oxidation–reduction processes and MFS transporters, were significantly up-regulated in phenamacril-resistant isolates. On the other hand, 14 DEGs involved in ATP-dependent RNA helicase and ribosomal biogenesis related proteins, showed significantly down-regulated expression in both phenamacril-resistant and -sensitive isolates. These results indicated that phenamacril not only seriously affected the cytoskeletal protein binding and ATPase activity of sensitive isolate, but also suppressed ribosome biogenesis in all the isolates. Hence, this study helps us better understand resistance regulation mechanism and fungicidal activity of phenamacril and provide reference for the development of new fungicides to control Fo.

Transcriptome Analysis of Fusarium-Tomato Interaction Based on an Updated Genome Annotation of Fusarium oxysporum f. sp. lycopersici Identifies Novel Effector Candidates That Suppress or Induce Cell Death in Nicotiana benthamiana.

Fusarium oxysporum f. sp. lycopersici (Fol) causes vascular wilt disease in tomato. Upon colonization of the host, Fol secretes many small effector proteins into the xylem sap to facilitate infection. Besides known SIX (secreted in xylem) proteins, the identity of additional effectors that contribute to Fol pathogenicity remains largely unexplored. We performed a deep RNA-sequencing analysis of Fol race 2-infected tomato, used the sequence data to annotate a published genome assembly generated via PacBio SMRT sequencing of the Fol race 2 reference strain Fol4287, and analysed the resulting transcriptome to identify Fol effector candidates among the newly annotated genes. We examined the Fol-infection expression profiles of all 13 SIX genes present in Fol race 2 and identified 27 new candidate effector genes that were likewise significantly upregulated upon Fol infection. Using Agrobacterium-mediated transformation, we tested the ability of 22 of the new candidate effector genes to suppress or induce cell death in leaves of Nicotiana benthamiana. One effector candidate designated Fol-EC19, encoding a secreted guanyl-specific ribonuclease, was found to trigger cell death and two effector candidates designated Fol-EC14 and Fol-EC20, encoding a glucanase and a secreted trypsin, respectively, were identified that can suppress Bax-mediated cell death. Remarkably, Fol-EC14 and Fol-EC20 were also found to suppress I-2/Avr2- and I/Avr1-mediated cell death. Using the yeast secretion trap screening system, we showed that these three biologically-active effector candidates each contain a functional signal peptide for protein secretion. Our findings provide a basis for further understanding the virulence functions of Fol effectors.

The Subunit Nto1 of the NuA3 Complex Is Associated with Conidiation, Oxidative Stress Response, and Pathogenicity in Fusarium oxysporum

Fusarium oxysporum f. sp. conglutinans (FOC) is the dominant pathogen of vascular wilt disease on cabbage and other crucifers. Foc-Nto1 was confirmed to be the homologous protein of Nto1, a subunit of the NuA3 (nucleosomal acetyltransferase of histone H3) complex in Saccharomyces cerevisiae. FOC contains two races, race 1 and race 2. The functions of Nto1 in both races were investigated through functional genetics analyses. The Nto1-deleted mutants were decreased in conidium production and displayed increased sensitivity to hydrogen peroxide. These mutants also had reduced virulence on cabbage. The study provided evidence that Nto1 is a potential metabolic- and pathogenic-related factor in F. oxysporum.

The Small Secreted Protein FoSsp1 Elicits Plant Defenses and Negatively Regulates Pathogenesis in Fusarium oxysporum f. sp. cubense (Foc4).

Fusarium wilt of banana (Musa spp.), a typical vascular wilt disease caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense race 4 (Foc4), seriously threatens banana production worldwide. Pathogens, including vascular wilt fungi, secrete small cysteine-rich proteins during colonization. Some of these proteins are required for pathogenicity. In this study, 106 small secretory proteins that contain a classic N-terminal signal peptide were identified using bioinformatic methods in Foc4. Among them, 11 proteins were selected to show transient expressions in tobacco. Interestingly, transient expression of FoSsp1 in tobacco, an uncharacterized protein (of 145 aa), induced necrotic cell death reactive oxygen burst, and callous deposition. Furthermore, the expression of FoSSP1 in Foc4 wild type (WT) was up-regulated during the stage of banana roots colonization. A split-marker approach was used to knock out FoSSP1 in the Foc4 WT strain. Compared with the WT, the deletion mutant Fossp1 was normal in growth rate but increased in conidiation and virulence. RT-qPCR analysis showed that the expression of four conidiation regulator genes in the Fossp1 deletion mutant was significantly decreased compared to the WT strain. In addition, the expression of four pathogenesis-related genes of bananas infected with Fossp1 deletion mutant was down-regulated in comparison with that of the WT. In summary, these results suggested that FoSSP1 is a putative elicitor that negatively regulates conidiation and pathogenicity in Foc4.

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

Summary 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.

Genomic regions linked to reniform nematode (Rotylenchulus reniformis) resistance in castor

Reniform nematode (Rotylenchulusreniformis) infects castor and makes it vulnerable to vascular wilt and root rot diseases. In this study, a population consisting of 92 recombinant inbred lines (RILs) derived from the cross between reniform nematode resistant line JC-12 and susceptible line 48-1 was used to identifythe genomic regions linked to reniform nematode resistance. The parents, F1 and RILs were screened against reniform nematode in pot culture with artificial inoculation of nematodes. The scoring for nematode resistance was done on the basis of number of nematodes extracted from the soil at 60 days after inoculation. The mean nematode count in 48-1 and JC-12 was 215.0±9.1 and 77.8±4.8 nematodes/ml of soil wash, respectively. The F1 reaction (217.3±13.2 nematodes/ml of soil wash) was similar to the susceptible parent indicating that nematode resistance in JC-12 is recessive in nature. The nematode count in RILs ranged from 43.8 to 327.3. QTL mapping using a linkage map consisting of 1,090 SNP markers resulted in the identification one QTLeach on chromosome-6 and chromosome-8, linked to resistance. This is the first report on mapping of genomic regions linked to reniform nematode resistance in castor, which form the basis for furthering the research on genetic and molecular biology of nematode resistance in castor.

RNAi-mediated down-regulation of fasciclin-like proteins (FoFLPs) in Fusarium oxysporum f. sp. lycopersici results in reduced pathogenicity and virulence

Fusarium oxysporum f. sp. lycopersici (Fol) is majorly responsible for causing vascular wilt disease in tomato by blocking transpirational pull, thereby interfering and suppressing overall host immune response. This is mainly achieved by fungal invasion and colonization within the host, resulting in hyphal formation that instigates secondary infection response inside the plant. Earlier reports show the role of fasciclin-like proteins (FLPs) in cell-to-cell adhesions and signaling cascade. Moreover, deletion mutant of FLPs explained its role in development stages of Magnaporthe oryzae and Lentula edodes. Therefore, in present study, based on bioinformatic analysis, we have identified putative FoFLP genes encoding Fusarium-specific fasciclin like proteins. We have exploited the RNAi technology to analyse and understand role of these FoFLPs in virulence during wilt disease in tomato. Interestingly, the morphogenesis of fungal RNAi transformants of FoFLP1, FoFLP3, FoFLP4 and FoFLP5 showed significant reduction in spore count and spore germination frequency, thereby suggesting role of FoFLPs in conidiation. Furthermore, we have detected FoFLP1, FoFLP3, FoFLP4 and FoFLP5 specific siRNAs in the respective fungal transformants, using stem-loop RT-PCR and northern hybridization suggesting targeting of cognate FoFLPs transcripts. Moreover, the fruit invasion and plant infection assays using FoFLP fungal transformants showed late onset of disease with significant reduction in disease symptoms in the infected plants. Although the FoFLP-RNAi fungal transformants entered the host plant by penetrating root cortex, the infected plants showed minimum fungal colonization as a result of siRNA-mediated targeting of endogenously expressed FoFLPs. This further inhibited the propagation of fungal RNAi transformants within root cortex and affected its ability to cause secondary infection response, which led to reduced disease incidence and disease severity index. Altogether, our results show that FoFLPs might play an important role in conidiation and pathogenicity, and can serve as potent RNAi targets. Therefore, Host-Induced Gene Silencing (HIGS) can effectively be used to target FoFLPs in order to raise tomato transgenics resistant to Fusarium wilt.

Identification and Functional Analysis of a Novel Hydrophobic Protein VdHP1 from Verticillium dahliae.

ABSTRACTVerticillium dahliae could cause destructive vascular wilt disease on hundreds of plant species around the world, including cotton. In this study, we characterized the function of a hydrophobin gene VdHP1 in pathogen development and pathogenicity. Results showed that VdHP1 could induce cell death and activate plant immune responses. The VdHP1 deletion mutants (ΔVdHP1) and the complement mutants (C-ΔVdHP1) were obtained by the homologous recombination method. The VdHP1 deletion mutants exhibited increased hydrophilicity, inhibited microsclerotial formation, and reduced spore smoothness. In addition, the deletion mutants were more sensitive to NaCl, while relatively insensitive to KCl and sorbitol. Mutants also had greater resistance to Congo red, UV radiation, and high temperature, which suggested that ΔVdHP1 strains have stronger resistance to abiotic stress in general. Different carbon source assays showed that the utilization ability of skim milk, cellulose, and starch was greatly enhanced in ΔVdHP1, compared with that of WT and complemented strains. Furthermore, VdHP1 did not affect mycelium penetration on cellophane but contributed to mycelium growth on surface of the living plant cells. The pathogenicity test found that the crude toxin content, colonization, and dispersal of ΔVdHP1 was significantly increased compared with the WT and complementary strains. In addition, cotton seedlings showed more severe wilting symptoms after inoculation with ΔVdHP1 strains. These results suggested that the hydrophobin VdHP1 negatively regulated the virulence of V. dahliae, and played an important role in development, adaptability, and pathogenicity in V. dahliae, which maybe provide a new viewpoint to further understand the molecular mechanisms of pathogen virulence.IMPORTANCE Verticillium dahliae is a soilborne fungal pathogen that causes a destructive vascular disease on a large number of plant hosts, resulting in great threat to agricultural production. In this study, it was illustrated that the hydrophobin VdHP1 could induce cell death and activate plant immune responses. VdHP1 affected the hydrophobicity of V. dahliae, and negatively regulated the strains resistant to stress, and the utilization ability of different carbon sources. In addition, VdHP1 did not affect mycelium penetration on cellophane but contributed to mycelium growth on surface of the living plant cells. The VdHP1 gene negatively regulated the total virulence, colonization, and dispersal of V. dahliae, with enhanced pathogenicity of mutant strains in this gene. These results suggested that the hydrophobin VdHP1 played an importance in development, adaptability, and pathogenicity in V. dahliae, and would provide a new viewpoint to further understand the molecular mechanisms of pathogen virulence.

Live-cell imaging elaborating epidermal invasion and vascular proliferation/colonization strategy of Verticillium dahliae in host plants.

The soilborne ascomycete fungus Verticillium dahliae causes destructive vascular wilt disease in hundreds of dicotyledonous plant species. However, our understanding of the early invasion from the epidermis to the vasculature and the prompt proliferation and colonization in the xylem tissues remains poor. To elaborate the detailed infection strategy of V. dahliae in host plants, we traced the whole infection process of V. dahliae by live‐cell imaging combined with high‐resolution scanning electron microscopy. The 4D image series demonstrated that the apex of invading hyphae becomes tapered and directly invades the intercellular space of root epidermal cells at the initial infection. Following successful epidermal invasion, the invading hyphae extend in the intercellular space of the root cortex toward the vascular tissues. Importantly, the high‐resolution microscopic and live‐cell images demonstrated (a) that conidia are formed via budding at the apex of the hyphae in the xylem vessels to promote systemic propagation vertically, and (b) that the hyphae freely cross adjacent xylem vessels through the intertracheary pits to achieve horizontal colonization. Our findings provide a solid cellular basis for future studies on both intracellular invasion and vascular colonization/proliferation during V. dahliae infection and pathogenesis in host plants.

Proteomic insights of chitosan mediated inhibition of Fusarium oxysporum f. sp. cucumerinum

Fusarium oxysporum f. sp. cucumerinum (FOC) infects cucumber plants, causing significant yield losses. Chitosan is a natural biodegradable compound that has antifungal properties. To understand the inhibitory mechanism of chitosan against FOC, a comprehensive proteomic study was carried out for the identification of chitosan responsive proteins (CRPs) from the mycelia of chitosan-treated FOC. Two-dimensional gel electrophoresis (2-DE) coupled with LC-MS/MS analysis led to the identification of 62 differentially abundant CRPs. Functional classification of these CRPs revealed that most proteins were involved in metabolism and defense. Gene Ontology analysis revealed that the majority of the proteins were assigned in proteolysis and hydrolase activity. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that among the biologically active pathways in chitosan-treated FOC mycelia, ‘carbohydrate metabolism’ was enriched for most of the proteins. This study gives a snapshot of the molecular basis of fungal inhibition by chitosan resulting in disease resistance in cucumber plants after inoculation with chitosan-treated FOC by explaining how chitosan restricted disease severity (i.e., down-regulating the plant cell wall degrading enzymes, FOC self-attack, hindering FOC structural and functional protein biosynthesis and DNA biosynthesis and affecting FOC transporter proteins). This study contributes to putting more weight on using the bioactive natural compound chitosan as an antifungal material instead of applying chemical fungicides in agriculture. SignificanceChitosan has been used as one of the safe and effective alternatives to fungicides in controlling cucumber vascular wilt disease caused by Fusarium oxysporum f. sp. cucumerinum (FOC) that is responsible for severe production losses. Chitosan application showed a significant decrease in wilt disease severity compared to chitosan untreated FOC and showed an efficiency of 91.7% in reducing pathogenicity. A comprehensive proteomic investigation of chitosan-responsive proteins (CRPs) from the mycelia of chitosan-treated FOC was carried out in order to better understand the inhibitory mechanism of chitosan against FOC which led us to identify 62 differentially expressed CRPs. Our proteomic study revealed CRPs in FOC involved in a variety of functions, including disease inhibition in cucumber. This study depicts what happens inside the fungus following treatment with chitosan and how chitosan played the role of the maestro in influencing the synthesis of proteins responsible for the virulence of FOC and their respective pathways, rendering FOC unable to infect the cucumber plant and lose its pathogenic potential to cause wilt disease. The efficiency of chitosan in inhibiting certain proteins or specific pathways of FOC gives a golden opportunity in controlling vascular wilt, so we highly recommend applying chitosan in disease management under greenhouse conditions or in the open field