The disease begins with dormant microsclerotia that are mixed in the soil or hiding in dead plant parts (Fig. 1). Root injuries often act as entry points for pathogens, facilitating the spread of infection. Under favorable conditions, the microsclerotia produce thin hyphae that extend towards the roots of nearby plants. There is limited evidence suggesting that hosts must meet specific requirements or interact in certain ways to restrict early root colonization during germination. Fungal growth is often observed between root surface cells, either near the root tips or after root hairs have emerged. However, most of these infections fail to reach the plant’s vascular system. These findings suggest that the specific location where initial establishment occurs, rather than the overall spread within the root system, plays a crucial role in facilitating efficient establishment of vascular invasion in the host organism. When vascular tissues are infected with Verticillium spp., conidia are formed in xylem vessels, and transported upwards through the transpiration stream. Conidia are usually trapped at the pit boundaries between vessels, which begins germination, penetrate the pit membrane to adjacent vessels, and proliferate, thereby initiating the infection cycle anew (Klosterman et al., 2009). The colonization of xylem vessels by pathogens disrupts sap flow, leading to vascular occlusion and impaired water transport, which ultimately results in the characteristic symptoms of wilt disease. In Chinese cabbage, symptoms typically appear approximately six weeks after transplanting. If symptoms develop before the heading stage, the outer leaves exhibit bright yellow discoloration accompanied by chlorosis (Fig. 2A). Conversely, if symptoms arise after the heading stage, the inner leaves become noticeably paler than the outer leaves, taking on a light green coloration (Fig. 2B). Infected plants display wilting and drooping of the outer leaves, while the inner leaves develop chlorosis and turn white on one side. Additionally, head formation is inhibited, resulting in poor development and stunted growth (Fig. 3A). Leaves lose their luster on one side of the midrib, followed by paling and wilting along the leaf veins. The margins turn bright yellow and eventually develop necrotic lesions (Fig. 3B). The roots also exhibit dark brown or black discoloration in the vascular tissue (Fig. 3C). Some studies suggest that the pathogen contributes to these symptoms by secreting a diverse array of toxins, including polysaccharides, protein-lipopolysaccharides, glycoproteins, and enzymes, some of which function as effectors that exacerbate wilting. As infected plant tissues decompose, microsclerotia are released into the soil, where they remain viable for several years, enabling the pathogen to persist and spread anew (De Sain and Rep, 2015; Klosterman et al., 2011; Kubicek et al., 2014; Pegg and Brady, 2002; Yadeta and Thomma, 2013; Zhang et al., 2022).

To determine the taxonomic status of the genus Verticillium, morphological and phylogenetic methods were employed. Verticillium comprises of plant-pathogenic species that are characterized as ascomycete filamentous fungi that commonly infect the vascular tissues of agricultural crops (Inderbitzin et al., 2011, 2013; Jeseničnik et al., 2023; Klosterman et al., 2009; Zare et al., 2007). The genus Verticillium was first identified in 1816 and is characterized by branched conidiophores forming whorls with flask-shaped phialides and end-point conidia. Since then, approximately 190 species have been documented, providing a strong foundation for its taxonomy (Klosterman et al., 2009; Pegg and Brady, 2002). Research studies have identified Verticillium dahliae and Verticillium longisporum as the primary causal agents of Verticillium wilt in Chinese cabbage (Dumin et al., 2020; Han et al., 2012; Horiuchi et al., 1990; Watanabe et al., 1973). Although V. dahliae has a broader host range and infects a larger number of plant species, V. longisporum is reported to be more destructive in Chinese cabbage, causing severe yield losses and increased disease severity (Ikeda et al., 2012). Prior to this, Kemmochi et al. (1999) reported that V. longisporum is more aggressive than V. dahliae in cabbage.

In 1912, the term “heterokaryosis” was introduced by Hans Burgeff and then the variability identification method was introduced based on compatibility groups (Burgeff, 1912; Krnjaja et al., 2013; Leslie, 1993). During the parasexual cycle, fungal hyphae, regardless of whether they are from the same or different species, can fuse through a process called anastomosis. The result of this fusion is the formation of a heterokaryon, which acts as the basis for vegetative compatibility. The formation of heterokaryons offers several potential advantages, including functional diploidy, genetic exchange during mitosis (parasexual cycle), and increased biomass to support cooperative physiological activities such as resource utilization and asexual or sexual reproduction. The alignment of specific genetic loci (het; heterokaryon incompatibility; also called vic for vegetative incompatibility) in each of them is essential for this process to occur (Glass et al., 2000; Klosterman et al., 2009; Leslie, 1993; Schardl and Craven, 2003; Strom and Bushley, 2016). Isolates with similar compatibility are grouped together into a Vegetative Compatibility Group (VCG), while those in different groups are considered genetically distinct populations, a process known as vegetative incompatibility, which is also sometimes referred to as heterogenic incompatibility, heterokaryon incompatibility, or somatic incompatibility (Glass et al., 2000). By pairing nitrate nonutilizing mutants (nits) on specific media, researchers identified various VCGs of the V. dahliae (Bhat and Subbarao, 1999; Subbarao et al., 1995). By testing a set of differential hosts, two V. dahliae isolates from Chinese cabbage were identified as pathotypes B (tomato pathotype) and C (pepper pathotype) (Horiuchi et al., 1990). Nitrate-nonutilizing mutants of these isolates were used to determine vegetative compatibility groups, classifying them as VCG J1 and VCG J2 (Japanese) subgroup respectively (Wakatabe et al., 1997). Complementation reactions between subgroup J1 isolates and VCG 2A/2B testers were performed later; this led to the proposal of assigning subgroup J1 to VCG 2A/B (Ebihara et al., 1999). Advancements in molecular techniques are enabling a more detailed exploration of diversity within V. dahliae populations, revealing intricate relationships between VCGs. The definite VCGs within V. dahliae may consist of genetically diverse isolates that are phylogenetically distant. While significant progress has been made in understanding the connections between V. dahliae and its diverse hosts, a critical need remains for more molecular markers. Applying these markers in a wider ecological context would be crucial for unraveling the origins and evolutionary diversification of V. dahliae and understanding how these factors influence the fungus’s biology (Jiménez-Gasco et al., 2014).

The usual life cycle of ascomycete fungi predominantly centers on the haploid stage. However, V. longisporum deviates from this norm as it is amphidiploid, resulting from hybridization of two haploid ancestors. This is why V. longisporum isolates from cabbage and cauliflower did not exhibit the nit mutant phenotype; however, they formed a distinct group based on random amplified polymorphic DNA (RAPD) patterns (Bhat and Subbarao, 1999). Sexual compatibility depends on the presence of opposite idiomorphs of the MAT locus, which is crucial for sexual recombination in ascomycetes. Most V. longisporum genomes contain copies of the MAT1-1 idiomorph, whereas MAT1-2 is prevalent in V. dahliae isolates. The unequal distribution and absence of a sexual cycle, along with significant sequence variations in MAT loci and distinct genetic clusters, suggest a limited potential for sexual reproduction in V. longisporum, making sexual propagation unlikely. Instead, hybridization is thought to occur through hyphal fusion, followed by nuclear fusion and genome duplication, leading to interspecific hybrids (Depotter et al., 2016a; Harting et al., 2021; Inderbitzin et al., 2011). Phylogenetic studies have classified V. longisporum isolates into three distinct lineages, each tracing back to different parental origins (Inderbitzin et al., 2011). If these hybrids become too adaptable, they might outcompete and entirely replace their parent strains, potentially driving those lineages to extinction. This scenario could explain the absence of observed parental lineages among V. longisporum (Depotter et al., 2016b). The parental origins of V. longisporum involve two genotypes from V. dahliae, specifically lineages D2 and D3, along with two unidentified species designated as Species A1 and Species D1. Analysis has shown that all identified V. longisporum isolates possess alleles inherited from the Species A1 progenitor, which combine with alleles from Species D1, V. dahliae lineage D2, or V. dahliae lineage D3 to generate the hybrid strains A1/D1, A1/D2, and A1/D3, respectively (Depotter et al., 2016a). A1/D3 and A1/D1 hybrid strains have been identified in Chinese cabbage, cabbage, oilseed rape, radish, and cauliflower across various regions, including Europe, China, Japan, and North America (Banno et al., 2014; Depotter et al., 2016a; Inderbitzin et al., 2011; Yu et al., 2015, 2016). Overall, V. longisporum exhibited greater virulence than V. dahliae on Brassicaceae hosts, while significant differences in virulence and pathogenicity observed across its lineages. The A1/D1 lineage is the most virulent across Brassicaceae crops, particularly affecting oilseed rape and cauliflower, while A1/D2 lineage is the most virulent on cabbage and horseradish. In contrast, A1/D3 lineage is generally less virulent (Novakazi et al., 2015; Tran et al., 2012).

In 1966, the Verticillium wilt was identified in Chinese cabbage in Nagano, Japan, with documented records dating back to 1973. The disease was initially attributed to Fusarium oxysporum; however, the pathogen was not found in diseased tissues. Instead, Verticillium albo-atrum was isolated (Watanabe et al., 1973). Subsequent RAPD analysis reclassified the pathogen as V. dahliae (Iijima, 1981; Ikeda et al., 2012; Koike et al., 1996). Later, the Verticillium wilt of Chinese cabbage was linked to diploid V. dahliae isolates, which were subsequently classified as Verticillium longisporum based on conidial size, restriction fragment length polymorphism (RFLP) analysis, and PCR techniques (Carder and Barbara, 1994; Horiuchi et al., 1990; Karapapa et al., 1997; Morton et al., 1995; Okoli et al., 1994). Following Japan, the fungus causing Verticillium wilt in Chinese cabbage in China was isolated and first identified as V. dahliae based on ribosomal DNA-ITS (internal transcribed spacer region) sequence analysis (Han et al., 2012). Further classification using mitochondrial small-subunit rDNA (mt-SSU rDNA) and cytochrome sequence alignment identified it as V. longisporum (Yu et al., 2015). In Korea, V. dahliae was identified in field-grown Chinese cabbages through morphological analysis and PCR of the ITS and RNA polymerase II (RPB2) gene regions (Dumin et al., 2020). Advancements in identification methods, such as PCR and sequencing, have greatly enhanced our understanding of the pathogen.

Conventional methods for identifying Verticillium spp., rely on morphological traits such as conidial length and shape, conidial and microsclerotial size, and enzymatic activity like polyphenol oxidase activity. These methods provide basic and effective approach for species differentiation. Findings indicate that V. dahliae isolates from Chinese cabbage exhibit shorter conidia, spherical and compact microsclerotia, and polyphenol oxidase activity. In contrast, V. longisporum isolates are characterized by longer conidia, irregularly shaped, elongated chain-like microsclerotia of varying sizes, and the absence of polyphenol oxidase activity (Ikeda et al., 2012; Yu et al., 2015).

However, conventional methods are labor-intensive, time-consuming, and often lack consistency across replicates. Moreover, they may struggle to distinguish Verticillium fungi from other fungi types in plant samples (Yu et al., 2015). Species differentiation was established between V. longisporum and V. dahliae in Chinese cabbage through 18S rDNA intron PCR, which showed the intron is present only in V. longisporum. In the field test, V. longisporum, exhibits more destructive symptoms compared to V. dahliae. Geographical variations in the distribution of these species were observed in Chinese cabbage-growing regions of Japan, emphasizing the need for region-specific management strategies (Ikeda et al., 2012). The A1/D1 strains of V. longisporum isolated from cabbage contain the group I 18S rDNA intron, distinguishing them from other lineages and the A1/D3 strains share the same 5.8S rDNA-ITS region as V. dahliae, indicating a closer genetic relationship between these strains and V. dahliae. However, these assays cannot differentiate the A1/D3 lineage of V. longisporum from V. dahliae. To address this, PCR-RFLP analysis was employed (Banno et al., 2014). In the PCR-RFLP analysis, the mt-SSU rDNA region and cytochrome b (cob) gene are amplified, providing another method to distinguish between these two species. Both methods, targeting different genetic regions, offer reliable differentiation V. dahliae and V. longisporum based on distinct DNA characteristics (Banno et al., 2014; Ikeda et al., 2012). To further investigate genetic variation between the A1/D1 and A1/D3 V. longisporum types, RAPD analysis, as described by Karapapa et al. (1997), was employed using Verticillium isolates from cabbage. Primers P4a and OPA13 generated polymorphic bands, with P4a amplifying a 1.4 kb band specific to V. longisporum and OPA13 producing 0.6 kb and 1.3 kb bands in all isolates, plus a 1.15 kb band unique to A1/D1-type. These results confirm V. longisporum as genetically distinct from V. dahliae and V. albo-atrum and highlight intraspecific variation, aiding its molecular identification (Banno et al., 2014).

PCR and gene sequencing facilitate species differentiation by generating unique genetic markers, which are subsequently used to construct phylogenetic trees based on sequence alignment (Yu et al., 2015). Advances in molecular techniques, sequencing-based methods, and taxonomic analyses of the ribosomal ITS, protein-coding genes actin (ACT), elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD), and tryptophan synthase and other DNA regions have enhanced our understanding of Verticillium spp., enabling more precise identification (Inderbitzin et al., 2011, 2013).

Various control strategies for Verticillium-induced diseases have been explored continuously. These include choosing appropriate planting locations, implementing quarantine restrictions, utilizing disease-free planting material, applying fungicides, employing soil fumigation, practicing crop rotation, adjusting soil fertility and irrigation practices, and opting for resistant cultivars (Cirulli et al., 1994; Pegg and Brady, 2002; Puri et al., 2021; Villani et al., 2021).

The extended viability of microsclerotia and their broad host range pose significant challenges to managing Verticillium wilt in various crops. As microsclerotia serve as the primary source of inoculum, reducing their presence in the soil is a key strategy for effective disease control. Methyl bromide was widely used for many years as a soil fumigant to manage Verticillium wilt until its complete discontinuation in 2005 due to significant environmental concerns. It was recognized as a major contributor to ozone depletion and posed risks to human health, including lung damage and neurological impacts. Alternative fumigants have been explored, like the combination of 1,3-dichloropropene and chloropicrin, dazomet, and metam sodium (Carroll et al., 2018; Duniway, 2002; Kowalska, 2021). Chloropicrin, dazomet, and metam sodium are commonly used in practical applications to manage the disease in Chinese cabbage. Shimizu et al. discovered that chloropicrin injection into plastic mulch ridges controlled Verticillium wilt in Chinese cabbage (Shimizu et al., 1983). In addition, chloropicrin tablets have been formulated to simplify application, demonstrating effectiveness in controlling Verticillium wilt in Chinese cabbage (Yoneyama and Tsukamoto, 1987). Ikeda et al. found that the Verticillium wilt potential of Chinese cabbage fields can be evaluated based on the disease incidence in other previous crops, the density of the root-lesion nematode that promotes disease occurrence, and the detection of the pathogen by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). This evaluation helps reduce unnecessary chloropicrin usage by applying it only to high-risk fields (Ikeda et al., 2014).

Chloropicrin fumigant is difficult to handle due to its strong odor. In contrast, dazomet granules produce less odor but require adequate soil moisture for effectiveness, making them less reliable in dry conditions (Fujinaga et al., 1999). Therefore, metam sodium (vapam) and metam ammonium were injected into a mulched ridge and sprayed prior to cultivation to control Verticillium wilt. This treatment effectively controlled the disease without visible phytotoxicity in fields in Japan (Fujinaga et al., 1999; Kobayashi et al., 1999; Watanabe et al., 2002). Using a tractor-mounted applicator reduced the time between pesticide application and soil incorporation, leading to consistent effectiveness. It was considered practical for field use due to its mild odor and ease of handling (Fujinaga et al., 1999; Watanabe et al., 2002). Although metam sodium and metam ammonium are somewhat less effective in the first year compared with chloropicrin soil disinfection, their effects persist in the soil for about two years and help control the disease. Additionally, by using disease-resistant varieties, it can serve as a more sustainable and effective approach for controlling Verticillium wilt (Watanabe et al., 2002). However, these substitutes have proven to be less effective than methyl bromide in managing the disease. Environmental problems related to chemicals are not hidden from anyone and have been mentioned many times in various articles, so experts looked for non-chemical alternative methods.

Ikeda and Osawa utilized species distribution modeling to demonstrate the likelihood of Verticillium wilt occurrence in cabbage monoculture. Considering these experiments and the threat of severe soil-borne diseases in Chinese cabbage, farmers can address these issues with different strategies. Soil disinfection is suitable for high-risk regions, whereas alternative methods can be used in low-risk areas (Ikeda and Osawa, 2020). Small pot-scale plants were utilized to create a dose-response curve (DRC) for predicting the presence of Verticillium wilt in Chinese cabbage, following the previously established protocol for clubroot diseases in cruciferous vegetables. By analyzing the slope values of the regression lines created from DRCs, researchers can predict how likely a particular Chinese cabbage field is to experience Verticillium wilt disease to assess the disease occurrence potential (D-potential) of fields. So, before cultivating the target fields, it is crucial to determine their D-potential to prevent excessive expenditures and to implement appropriate restrictions (Yoshida et al., 2021).

Biocontrol agent (BCA) involves applying beneficial microorganisms to manage plant diseases like Verticillium wilt. Soil suppressiveness is influenced by biotic interaction such as competition, antibiosis, plant resistance enhancement, and (hyper)parasitism between several bacterial and fungal. It was demonstrated that the combination of endophytic bacteria (Pseudomonas sp.) inoculated seedlings and urea polymer soil mixing showed some potential in controlling Verticillium wilt in Chinese cabbage, but the effectiveness was weaker in fields with high disease occurrence (Watanabe et al., 2003).

During a comprehensive screening for endophytic fungi with BCA capabilities, it was identified that dark septate endophytic fungus including Phialocephala fortinii, Heteroconium chaetospira, and Meliniomyces variabilis (LtVB3) have the ability to reduce symptoms caused by V. longisporum in Chinese cabbage grown in vitro when the plants were colonized by the BCA prior to infection. However, only isolates of H. chaetospira and M. variabilis effectively control the disease severity of Verticillium wilt in Chinese cabbage in natural field conditions. these root endophytic fungi are known to thrive on Chinese cabbage roots and inhibit the colonization of Verticillium wilt and trigger strong antagonistic responses in host plants, a phenomenon that can provide disease resistance (Depotter et al., 2016a; Narisawa et al., 2000, 2004; Ohtaka and Narisawa, 2007). Additionally, H. chaetospira has been found to form a mutualistic symbiosis with Chinese cabbage, facilitating nitrogen supply in exchange for carbohydrates and thereby promoting plant growth (Usuki and Narisawa, 2017).

Researchers in Japan have conducted comparative studies on microbial population in field soils with varying disease severity to understand the factors associated with disease suppression in Chinese cabbage Verticillium wilt. They used PCR-DGGE to assess these populations and found that the DGGE band was tightly associated with sequences belonging to the Chaetomiaceae family, which includes numerous genera of saprobic ascomycetes like Chaetomium, Guanomyces, and Thielavia and then they isolated fungal strain S69 (Noguchi et al., 2020). Under the updated taxonomic framework, utilizing both phylogenetic analysis and morphological examinations, scientists pinpointed Pseudothielavia terricola, as the fungal strain S69, as the agent responsible for combating Chinese cabbage Verticillium wilt (Noguchi et al., 2022). However, this association alone is insufficient for suppressing Chinese cabbage infection. Deeper exploration is necessary to recognize other contributing aspects and gain an improved insight into the mechanisms of soil suppressiveness.

In China, three Chinese cabbage lines were completely resistant to Verticillium wilt, having adapted to saline-alkaline soils and a temperate marine climate. Disease resistance patterns varied among Chinese cabbage types, aiding targeted germplasm screening. Controlled experimental inoculation is crucial for accurate resistance evaluation (Su et al., 2018).

Scientists combined a genome-wide association investigation conducted within a native population with quantitative trait locus mapping in an F2 population of Chinese cabbage to investigate the role of the MYB transcription factor BrMYB108 in modulating plant resistance to Verticillium wilt. Experiments on disease resistance revealed that high expression of BrMYB108 is linked to increased resistance to V. longisporum. Furthermore, it was discovered that BrMYB108 helps resist V. longisporum by controlling reactive oxygen species (ROS) production through its interaction with respiratory burst oxidase genes (Rboh) promoters. So, BrMYB108 and its target ROS genes could serve as promising candidate for genetic engineering aimed at enhancing V. longisporum resistance in Brassica rapa. However, BrMYB108 alone is not sufficient for resistance against Verticillium wilt, as resistance is regulated by multiple genes and complex networks. In addition, while BrMYB108 restricts V. longisporum spread in the shoot base, it does not actively defend against initial root infection, allowing early pathogen establishment. This limitation highlights the need for a broader strategy that strengthens root-specific defense mechanisms to enhance resistance in Chinese cabbage (Su et al., 2023).

Verticillium wilt poses a serious threat to Chinese cabbage cultivation and is primarily caused by V. longisporum and V. dahliae. Based on published studies and unpublished data, V. longisporum exhibits higher incidence and pathogenicity in Chinese cabbage compared to V. dahliae. These pathogens infect the plant’s vascular system, leading to wilting, yellowing, and eventual death, which severely impacts crop yields. Therefore, accurate detection and identification are crucial for effective disease management.

Advancements in diagnostic techniques, particulary molecular approaches, have significantly improved our ability to detect and quantify Verticillium in Chinese cabbage plants, even in field-contaminated environments. Early detection and identification is crucial, as it enables the rapid implementation of control methods. The real-time PCR technique was developed using the primer pair derived from the ITS1 and ITS2 regions and accurately quantified V. longisporum in both inoculated and field-contaminated plants in China. These attributes make it an effective tool for large-scale screening and early detection of Verticillium wilt in breeding programs (Yu et al., 2015). Additionally, quantitative nested real-time PCR and PCR-DGGE have been successfully used to detect V. dahliae and V. longisporum in field soils, demonstrating their potential for Chinese cabbage fields by revealing the presence, prevalence, and frequency of Verticillium species in infected soil samples (Banno et al., 2011; Ikeda et al., 2014). The presence of Verticillium is often a precursor to significant disease outbreaks in subsequent growing seasons, emphasizing the importance of regular soil health monitoring in areas with a history of Verticillium wilt.

While advances in detection have provided valuable tools for detecting Verticillium infections, effective disease control remains a major challenge. Further research is needed to explore the genetic basis of Verticillium pathogenicity, as well as the complex interactions between the pathogen and its host plant. A clearer understanding of these interactions will enable the development of more effective control strategies that can be incorporated into sustainable agriculture practices. In general, chemical approaches, such as methyl bromide and chloropicrin, are considered the most effective for controlling the disease, but their environmental impact cannot be ignored. Cultural practices like crop rotation and fertilization can help reduce the incidence and severity of Verticillium wilt but are not entirely reliable on their own. Biological control agents and host resistance have also been explored, though their effectiveness varies depending on environmental conditions and pathogen specificity. Therefore, an integrated approach may be necessary for more effective disease management.

It was confirmed that nematode plays a significant role in promoting Verticillium wilt in Chinese cabbage. The Shannon-Wiener diversity index for nematodes, measured using PCR-DGGE in Chinese cabbage field soils after harvest, was negatively linked to disease severity of plants grown in the next year (Momota et al., 1989; Nagase et al., 2015; Noguchi et al., 2020). The application of nematicides has been shown to reduce disease incidence in affected fields, with varying effectiveness depending on the type of pesticide used. However, mixed results from additional trials suggest that factors such as nematode population density and other environmental conditions must be considered. Further investigation into the specific conditions affecting nematode presence and disease development is needed for more reliable control methods (Ishizaka et al., 1988).

In summary, Verticillium wilt continues to pose a significant challenge to Chinese cabbage cultivation, but the combination of advanced diagnostic methods, cultural practices, and ongoing research into the genetic research offers hope for more effective disease management. By integrating molecular techniques, such as real-time PCR, with sustainable farming practices, it is possible to reduce the impact of Verticillium infections on Chinese cabbage crops and improve overall crop resilience. Future research focused on the pathogen-host interaction and the development of resistant cultivars will be pivotal in ensuring long-term solutions for Verticillium wilt control.