Dynamic infection of Verticillium dahliae in upland cotton

• Verticillium wilt, an infection caused by the soilborne fungus Verticillium dahliae, is one of the most serious diseases in cotton. No effective control method against V. dahliae has been established, and the infection mechanism of V. dahliae in upland cotton remains unknown.
• GFP‐tagged V. dahliae isolates with different pathogenic abilities were used to analyse the colonisation and infection of V. dahliae in the roots and leaves of different upland cotton cultivars, the relationships among infection processes, the immune responses and the resistance ability of different cultivars against V. dahliae.
• Here, we report a new infection model for V. dahliae in upland cotton plants. V. dahliae can colonise and infect any organ of upland cotton plants and then spread to the entire plant from the infected organ through the surface and interior of the organ.
• Vascular tissue was found to not be the sole transmission route of V. dahliae in cotton plants. In addition, the rate of infection of a V. dahliae isolate with strong pathogenicity was notably faster than that of an isolate with weak pathogenicity. The resistance of upland cotton to Verticillium wilt was related to the degree of the immune response induced in plants infected with V. dahliae. These results provide a theoretical basis for studying the mechanism underlying the interaction between V. dahliae and upland cotton. These results provide a theoretical basis for studying the mechanism underlying the interaction between V. dahliae and upland cotton.

     

Effects of cotton–maize rotation on soil microbiome structure

Verticillium wilt is a disastrous disease in cotton-growing regions in China. As a common management method, cotton rotation with cereal crops is used to minimize the loss caused by Verticillium dahliae. However, the correlation between soil microbiome and the control of Verticillium wilt under a crop rotation system is unclear. Therefore, three cropping systems (fallow, cotton continuous cropping, and cotton–maize rotation) were designed and applied for three generations under greenhouse conditions to investigate the different responses of the soil microbial community. The soil used in this study was taken from a long-term cotton continuous cropping field and inoculated with V. dahliae before use. Our results showed that the diversity of the soil bacterial community was increased under cotton–maize rotation, while the diversity of the fungal community was obviously decreased. Meanwhile, the structure and composition of the bacterial communities were similar even under the different cropping systems, but they differed in the soil fungal communities. Through microbial network interaction analysis, we found that Verticillium interacted with 17 bacterial genera, among which Terrabacter had the highest correlation with Verticillium. Furthermore, eight fungal and eight bacterial species were significantly correlated with V. dahliae. Collectively, this work aimed to study the interactions among V. dahliae, the soil microbiome, and plant hosts, and elucidate the relationship between crop rotation and soil microbiome, providing a new theoretical basis to screen the biological agents that may contribute to Verticillium wilt control.     

VdSho1 Regulates Growth,Oxidant Adaptation and Virulence in Verticillium dahliae

Verticillium dahliae infection leads to Verticillium wilt in cotton and other dicotyledon crops. To reduce the loss of economic crops, more attention has been focused on the key genes involved in pathogenicity of this soil‐borne plant fungal pathogen. Sho1 encodes a conserved tetraspan transmembrane protein which is a key element of the two upstream branches of the HOG‐MAPK pathway in fungi. Sho1 is required for full virulence in a wide variety of pathogenic fungi. In this study, sho1 mutant in V. dahliae (designated ΔVdsho1) was generated by Agrobacterium tumefaciens‐mediated transformation. ΔVdsho1 strain was highly sensitive to menadione (at concentration of 120 μm ) and hydrogen peroxide (at concentration of 250 μm ), displayed delayed spore germination and reduced spore production compared with the wild type and the complemented strains. During infection of host cotton plants, ΔVdsho1 exhibited impaired ability of root attachment and invasive growth. Results from the present work suggest that VdSho1 controls external sensing, virulence and multiple growth‐related traits in V. dahliae and might serve as a potential target for control of Verticillium wilt.     

Genetic transformation of cotton with a harpin-encoding gene <Emphasis Type="Italic">hpa</Emphasis><Subscript><Emphasis Type="Italic">Xoo</Emphasis></Subscript> confers an enhanced defense response against different pathogens through a priming mechanism

Background  

The soil-borne fungal pathogen Verticillium dahliae Kleb causes Verticillium wilt in a wide range of crops including cotton (Gossypium hirsutum). To date, most upland cotton varieties are susceptible to V. dahliae and the breeding for cotton varieties with the resistance to Verticillium wilt has not been successful.     

The island cotton NBS‐LRR gene GbaNA1 confers resistance to the non‐race 1 Verticillium dahliae isolate Vd991

Wilt caused by Verticillium dahliae significantly reduces cotton yields, as host resistance in commercially cultivated Gossypium species is lacking. Understanding the molecular basis of disease resistance in non‐commercial Gossypium species could galvanize the development of Verticillium wilt resistance in cultivated species. Nucleotide‐binding site leucine‐rich repeat (NBS‐LRR) proteins play a central role in plant defence against pathogens. In this study, we focused on the relationship between a locus enriched with eight NBS‐LRR genes and Verticillium wilt resistance in G. barbadense. Independent virus‐induced gene silencing of each of the eight NBS‐LRR genes in G. barbadense cultivar Hai 7124 revealed that silencing of GbaNA1 alone compromised the resistance of G. barbadense to V. dahliae isolate Vd991. In cultivar Hai 7124, GbaNA1 could be induced by V. dahliae isolate Vd991 and by ethylene, jasmonic acid and salicylic acid. Nuclear protein localization of GbaNA1 was demonstrated by transient expression. Sequencing of the GbaNA1 orthologue in nine G. hirsutum accessions revealed that all carried a non‐functional allele, caused by a premature peptide truncation. In addition, all 10 G. barbadense and nine G. hirsutum accessions tested carried a full‐length (～1140 amino acids) homologue of the V. dahliae race 1 resistance gene Gbve1, although some sequence polymorphisms were observed. Verticillium dahliae Vd991 is a non‐race 1 isolate that lacks the Ave1 gene. Thus, the resistance imparted by GbaNA1 appears to be mediated by a mechanism distinct from recognition of the fungal effector Ave1.     

Effect of Inoculum Density on Verticillium Wilt Incidence in Commercial Olive Orchards

Verticillium wilt, caused by Verticillium dahliae Kleb., is presently the most destructive disease of olive, particularly in Andalucía (southern Spain). ‘Picual’ and ‘Arbequina’ are the dominant cultivars being planted in Spain. Both cultivars are highly susceptible to the defoliating pathotype of V. dahliae when artificially inoculated by root‐dipping or stem injection. Conversely, ‘Arbequina’ is considered more resistant than ‘Picual’ based on field observations and farmer's experience. In this study, the differential reaction between of cultivars was confirmed by surveys of naturally infested orchards with different inoculum densities of the pathogen. The average percentage of affected olive trees of ‘Picual’ was 60.2%, while only 13.1% of trees of ‘Arbequina’ showed disease symptoms. Overall, the pathogen caused extensive wilting of branches and defoliation on the trees of ‘Picual’, whereas ‘Arbequina’‐infected trees showed chlorotic symptoms and slight defoliation. The relationship between inoculum density and disease incidence fit a logarithmic function for both cultivars. The percentage of affected trees of ‘Arbequina’ per year increased linearly (y = 0.3559x, R² = 0.5652, and P = 0.0195) with the inoculum density in the soil, whereas this relationship was not observed for the ‘Picual’. Planting density had no effect on disease incidence for any of the two cultivars.     

Island Cotton Gbve1 Gene Encoding A Receptor-Like Protein Confers Resistance to Both Defoliating and Non-Defoliating Isolates of Verticillium dahliae

Verticillium wilt caused by soilborne fungus Verticillium dahliae could significantly reduce cotton yield. Here, we cloned a tomato Ve homologous gene, Gbve1, from an island cotton cultivar that is resistant to Verticillium wilt. We found that the Gbve1 gene was induced by V. dahliae and by phytohormones salicylic acid, jasmonic acid, and ethylene, but not by abscisic acid. The induction of Gbve1 in resistant cotton was quicker and stronger than in Verticillium-susceptible upland cotton following V. dahliae inoculation. Gbve1 promoter-driving GUS activity was found exclusively in the vascular bundles of roots and stems of transgenic Arabidopsis. Virus-induced silencing of endogenous genes in resistant cotton via targeting a fragment of the Gbve1 gene compromised cotton resistance to V. dahliae. Furthermore, we transformed the Gbve1 gene into Arabidopsis and upland cotton through Agrobacterium-mediated transformation. Overexpression of the Gbve1 gene endowed transgenic Arabidopsis and upland cotton with resistance to high aggressive defoliating and non-defoliating isolates of V. dahliae. And HR-mimic cell death was observed in the transgenic Arabidopsis. Our results demonstrate that the Gbve1 gene is responsible for resistance to V. dahliae in island cotton and can be used for breeding cotton varieties that are resistant to Verticillium wilt.     

Phylogenetic analysis of <Emphasis Type="Italic">Verticillium</Emphasis> species based on nuclear and mitochondrial sequences

Three different genes were sequenced from isolates of five plant-pathogenic Verticillium species, Verticillium albo-atrum, Verticillium dahliae, Verticillium longisporum, Verticillium nigrescens, and Verticillium tricorpus. The sequences covered parts of the mitochondrial cytochrome b gene (cob), the mitochondrial small subunit rRNA gene (rns) and the nuclear ITS2 region. When the sequences were combined, the five species clustered in five monophyletic groups, with V. nigrescens distantly related to the other species while V. tricorpus displayed a somewhat closer relationship to the three remaining species. V. albo-atrum, V. dahliae and V. longisporum were found to be very similar to each other, with V. albo-atrum and V. longisporum displaying the closest relationship. The species affiliation of V. longisporum is discussed.     

Colonization of Olive Inflorescences by Verticillium dahliae and its Significance for Pathogen Spread

Verticillium wilt of olive, caused by Verticillium dahliae Kleb., is the most severe disease affecting this crop in most olive growing countries. In this study, the presence of viable structures of V. dahliae in dried inflorescences from wilted olive shoots was investigated. The pathogen was found inside peduncles and flowers, by assessing the number of typical star‐shaped microsclerotial colonies formed onto the modified sodium polypectate agar medium. Microsclerotia of V. dahliae were observed inside the peduncles under the stereoscopic microscope. The presence of microsclerotia in these easily decomposable olive tissues shows that infected inflorescences can act as a source of inoculum for Verticillium wilt epidemics.     

Reaction of Cotton Cultivars and an F2 Population to Stem Inoculation with Isolates Verticillium dahliae

Four Verticillium dahliae isolates (V76, TS‐2, PH, and V44) were used in screening four cotton cultivars (Pima S‐7, Acala Prema, M‐315 and Acala 44). Pima S‐7 and Acala Prema gave the highest resistance reactions and Acala 44 was the most susceptible. Isolate V76 of V. dahliae was the most virulent. An interspecific cross between the resistant cv Pima S‐7 (Gossypium barbadense) and the susceptible cv. Acala 44 (G. hirsutum) was made and the F₂ population phenotyped for Verticillium wilt effect. Phenotyping of plant reaction to the disease was quantified by using a set of six growth parameters (number of healthy leaves, number of nodes, leaf weight, stem weight, leaf to stem ratio, and total shoot weight) measured 3 weeks after inoculation. The F₂ phenotypic distribution of these parameters suggests that distribution is towards resistance and polygenic. Transgressive segregation also was observed. The number of healthy leaves and total shoot weight were found to be the best indicators of resistance. Results obtained in this study will be useful to quantify resistance to V. dahliae and identify the best parameters to phenotype in genetic studies.     

Host Specialization among Vegetative Compatibility Groups of Verticillium dahliae in Relation to Verticillium longisporum

A collection of 24 isolates of Verticillium dahliae and 10 isolates of Verticillium longisporum originating from nine different host plants and from several geographic regions was tested for host specificity on 11 economically important crops such as potato, tomato, strawberry, linseed, three legumes and four Brassica species. In order to reveal host specificity the potential of each isolate to induce disease and affect plant yield was recorded for all isolate–host combinations. The collected data were statistically processed by means of a cluster analysis. As a result, the host range of individual isolates was found to be more dependent on the vegetative compatibility group (VCG) of the isolate than on its original host plant provenance. Twenty‐two out of 24 V. dahliae isolates belonged to either VCG 2B or 4B. VCG 2B isolates showed specificity for legumes, strawberry, potato and linseed, whereas VCG 4B was specifically virulent on potato, strawberry and linseed. Subgroups within VCG 2B and 4B almost lacking any host preference were designated 2B* and 4B*. Three isolates from VCG 2B*, however, severely attacked tomato which is a host outside the authentic host range of VCG 2B. The pathogenicity of V. longisporum isolates was restricted to cruciferous hosts. Conversely, cruciferous plants were not affected by isolates from VCGs 2B and 4B of V. dahliae. This lack of cross‐infectivity of certain subpopulations of V. dahliae and of V. longisporum may be useful in the management of this soil‐borne wilt disease.     

Genome‐wide association study discovered candidate genes of Verticillium wilt resistance in upland cotton (Gossypium hirsutum L.)

Verticillium wilt (VW), caused by infection by Verticillium dahliae, is considered one of the most yield‐limiting diseases in cotton. To examine the genetic architecture of cotton VW resistance, we performed a genome‐wide association study (GWAS) using a panel of 299 accessions and 85 630 single nucleotide polymorphisms (SNPs) detected using the specific‐locus amplified fragment sequencing (SLAF‐seq) approach. Trait–SNP association analysis detected a total of 17 significant SNPs at P < 1.17 × 10^–5 (P = 1/85 630, –log₁₀P = 4.93); the peaks of SNPs associated with VW resistance on A10 were continuous and common in three environments (RDIG2015, RDIF2015 and RDIF2016). Haplotype block structure analysis predicted 22 candidate genes for VW resistance based on A10_99672586 with a minimum P‐value (–log₁₀P = 6.21). One of these genes (CG02) was near the significant SNP A10_99672586 (0.26 Mb), located in a 372‐kb haplotype block, and its Arabidopsis AT3G25510 homologues contain TIR‐NBS‐LRR domains that may be involved in disease resistance response. Real‐time quantitative PCR and virus‐induced gene silencing (VIGS) analysis showed that CG02 was specific to up‐regulation in the resistant (R) genotype Zhongzhimian2 (ZZM2) and that silenced plants were more susceptible to V. dahliae. These results indicate that CG02 is likely the candidate gene for resistance against V. dahliae in cotton. The identified locus or gene may serve as a promising target for genetic engineering and selection for improving resistance to VW in cotton.     

Isolation of microsatellite markers from an interspecific hybrid isolate of the fungal plant pathogen Verticillium dahliae

Ten microsatellite markers have been isolated from an amphihaploid isolate of Verticillium dahliae using genomic libraries enriched for (AGT), (GAC), (GCC), (TAC) and (TTA) repetitive elements. These were characterized on four amphihaploid isolates and two haploid isolates each of V. dahliae and Verticillium albo‐atrum. Nine were polymorphic, with between two and five alleles, and the tenth was polymorphic when tested on a further 20 haploid V. dahliae isolates (three alleles). Only one primer pair gave the expected double bands from the amphihaploid isolates, supporting the view that V. albo‐atrum is not one of the parents of the interspecific hybrid amphihaploid isolates.     

Genetic relationships between virulence,vegetative compatibility and ISSR marker of Verticillium dahliae isolated from cotton

Verticillium dahliae is one of the most important pathogens causing Verticillium wilt. We studied the characterisation of the genetic relationship between virulence, vegetative compatibility groups (VCGs) and inter-simple sequence repeat (ISSR). A total of 48 V. dahliae isolates, in which 16 are collected from different cotton growing regions in China and 4 isolates belonged to all known VCGs, are used. Half of them were found highly virulent. Mutants (565) were obtained using the nitrate non-utilising mutant. These mutants were grouped into three VCGs: VCG1 (27 isolates), VCG 2 (14 isolates) and VCG 3 (7 isolates). Use of ISSR indicated two main clusters that were related to VCG and virulence. Genetic diversity lineages were obviously correlated to VCGs and ISSRs according to their geographical origin, virulence and ISSR genetic variation. This study could be useful to design and develop effective management strategies beside for quarantine purposes on Verticillium wilt control.