UNVEILING THE CLUBROOT PATHOGEN PLASMODIOPHORA BRASSICAE: INSIGHTS INTO ITS BIOLOGY, PATHOGENICITY, AND CONTROL STRATEGIES

Clubroot is a disaster in the cultivation of crops of the Cruciferae family, caused by an obligate fungus, ( Plasmodiophora brassicae ). This pathogen survives in soil and crop debris for a long time in the form of a double-walled resting spore which is sub spherical to spherical in shape with 3 µm in diameter. Its severity is highest at a pH of 5.7, a cool temperature, and excess moisture. The biotic factors in its severity include the spore count in the soil and its virulence. Its dominant resting spore germinates to produce primary plasmodia. The primary plasmodia infect root cells, producing zoospores, which infect cortical cells and cause hypertrophy. This causes formation of typical club shaped galls in the roots. For its management, an integrated management system of agronomic, biological, and chemical approaches is required. Agronomic strategies include liming to raise pH, Boron application, crop rotation, cultivating resistant varieties, soil solarization, and sanitation. Similarly, biological strategies include use of microbial organisms like Trichoderma spp., Gliocladium catenulatum, Streptomyces sp., Bacillus amyloliquefaciens, and endophytes like Acremonium alternatum and Heteroconium chaetospira. Finally, chemical approach includes the use of fungicides like cyazofamid, Penta Chloro Nitro Benzene (PCNB), Nano Silver Hydrogen Peroxide

the required amount of pure DNA is very difficult to retrieve (Stjelja et al., 2019). (Schwelm et al., 2015) published the genome of clubroot pathogen for the first time in history from the genomic DNA of resting spores of a single spore isolate e3, a European isolate (Javed et al., 2022). The genome of P. brassicae was found to be 24 Mb with the prediction of 9730 genes which had the support of seven transcriptome libraries and contained 4 introns per gene of length 60 base pairs (bp). The complete genome of e3 was published by (Stjelja et al., 2019) with the total size of 25.1 Mb which has helped a lot in the comparative study of genomics of Plasmodiophorids. Along with this isolate, five Canadian isolates (Rolfe et al., 2016) and a Chinese isolate (Bi et al., 2016) have been available for the study. The pathotype classification of these isolates is not easy due to their different differential hosts sets (Daval et al., 2019).
The genome of P. brassicae contains genes which could be potentially associated with the manipulation of host hormones such as isopentenyl-transferases, methyltransferase and cytokinin oxidase, and auxin-responsive Gretchen Hagen 3 (Schwelm et al., 2015). Like other eukaryotic biotrophic plant pathogens, P. brassicae also lacks several metabolic pathways (Daval et al., 2019). The missing genes in this pathogen encode proteins associated with arginine, thymine, lysine, and fatty acids biosynthesis pathways along with the proteins involved in sulfur and nitrogen uptake. Moreover, only few enzymes that are involved in the synthesis, transportation, and metabolism of carbohydrates are found in P. brassicae (Daval et al., 2019). Finally, the characterization of the P. brassicae is incomplete due to the limited number of genotypes and conditions from where above data are taken (Daval et al., 2019).
In 2009, the first clubroot-resistant (CR) canola cultivar became available to farmers, along with other cultivars from various seed companies. Genetic resistance has become the most important tool in Canada for managing clubroot, with 40 registered CR cultivars currently accessible. (Canola encyclopedia). Genetic resistance depends on single major genes that are efficacious against specific pathotypes or races of P. brassicae. (Rahman et al., 2014). A study also made by (Javed et al., 2022) focused on pathotyping, concluded that P. brassicae can be divided into pathotypes or races based on their prevailing virulence on divergent hosts. To understand the interrelation between P. brassicae and its hosts, five main pathotyping systems have been established. Currently, the Canadian clubroot differential, which involves a set of 13 hosts, is being used and has revealed 36 different pathotypes so far.
For proper clubroot identification, it is important to rapidly distinguish between different pathotypes, especially with the emergence of new pathotypes that can overcome resistance. Therefore, a different diagnostic approach is necessary for detecting pathotypes in clubroot. Currently, in bioassays with host differential sets, the responses of the hosts are observed based on the development of root galls, and P. brassicae pathotypes are phenotypically distinguished based on their virulence patterns. These tests are designed to measure the prevalence and degree of physiologic specialization in pathogen populations (Fredua-Agyeman et al., 2018). Apart from phenotypic approaches, microscopy also plays a role in pathotype identification. Various staining methods have been used under microscopy to observe the structure of P. brassicae and the host. Among these methods, the triple staining method is used to differentiate resting spores. (Buczacki & Moxham, 1979). Microscopy methods are ineffective for pathotyping because the cell morphology remains identical across different pathotypes. However, a PCR assay was developed for the detection of P. brassicae in soil. This assay is useful for identifying the presence of the pathogen and provides a more reliable and sensitive approach for pathotyping. (Ito et al., 1999). The primers used in the PCR assay were based on an isopentenyltransferase-like gene that is specific to P. brassicae. Additionally, another PCR assay was developed, which targets the internal transcribed spacer (ITS) region of ribosomal DNA, aiming to enhance sensitivity in pathogen detection (Faggian et al., 2007).
A different approach, known as metabarcoding, was studied for the detection of pathotypes present in P. brassicae. Metabarcoding utilizes Next Generation Sequencing (NGS), which provides high-quality single nucleotide resolution in a single reaction (Taberlet et al., 2012). Metabarcoding works by extracting DNA from the sample, which is then subjected to an initial PCR to generate barcoded amplicons. These amplicons are subsequently prepared for next-generation sequencing. To identify the pathotypes present in the sample, the sequencing reads are aligned to the reference barcode database (Tso et al., 2021b). Hence, overall, molecular diagnostics approaches are considered fundamental for detecting pathotypes due to their high sensitivity, rapidity, and cost-effectiveness in terms of labor, space, and time. Additionally, these approaches do not pose biosecurity concerns and overcome the limitation of inter-rater reliability (Tso et al., 2021a).
Pathogenicity is the qualitative capacity of a parasite to infect and cause disease on a host, and virulence is the degree of damage caused to a host by parasitic infections, which is thought to be negatively correlated with host fitness (SACRISTÁN & GARCÍA-ARENAL, 2008). Because it maintains evolving new pathotypes and changes in the distribution and frequency of previously existing pathotypes within individual fields, the P. brassicae pathogen is remarkably variable in virulence. As a result, changes in the P. brassicae pathotype can overcome resistance in previously clubroot-resistant cultivars, resulting in unexpected disease outbreaks and additional yield losses (Zamani-Noor, 2017). P. brassicae isolates' virulence and physiologic specialization were first recognized in the 1930s (Honig, 1931b).
A research assessment of Clubroot disease severity collected from different Location found that disease incidence and disease severity percentage were significantly influenced by different pathotypes collected in a study of inoculated samples of cauliflower with different inoculum collected from different locations. At 65 DAI, seedling inoculated with inoculum collected from Kavre, Dhading, and Makwanpur districts had the highest disease incidence (100%), while seedling inoculated with Lalitpur had the lowest disease incidence (50%) (Ghimire et al., 2022).
Another experiment done on a series of greenhouse condition in order to assess the effect of Plasmodiophora brassicae virulence on clubroot development and resting spore propagation in 86 plant species from 19 botanical families concluded that P1 (+)-inoculated species had more severe symptoms (two to ten times more severe), larger galls (1.1 to 5.8 times heavier), and more resting spores than P1-inoculated plants. Hence, the emergence and spread of new virulence pathotypes of P. brassica capable of overcoming resistance highlights the significance of plant species selection in farming systems in clubroot-infected field (Zamani-Noor et al., 2022).
For the study of the life cycle of P. brassicae, several attempts were made to culture it on artificial media, but were not successful; however, the callus culture technique was used to study the life cycle of this pathogen where callus was generated from an infected root tissue of Brassica spp. The life cycle of P. brassicae is divided into three stages viz. resting spore stage, primary stage and secondary stage (Ingram & Tommerup, 1972). The resting spores are the primary inoculums, are sub-spherical to spherical in shape, and are about 3 µm in size (Kageyama & Asano, 2009). The dormant resting spores have refractile globules which might contain some materials for storage that is mobilized as the germination process of the spores starts (Macfarlane, 1970). The refractile globules dissolve and disappear after the onset of germination process where the dissolution starts from one side and spreads across the entire spore (Macfarlane, 1970). This paper finds out the presence of a small papilla which emerges from a pore in the spore wall of the resting spore; however, the relationship between the place where globules start to disappear and the formation of pore in the spore wall is undetermined. The spores do not germinate in in-vitro conditions below 20 o C but when seedlings are provided, they germinate in them, probably because seedlings provide necessary conditions for the spores to germinate and compensate for the low temperature (Chupp, 1917). The optimum temperature for the spores germination in in-vitro condition is 25 o C (Wellman, 1930).
After the resting spore germination, the primary zoospores, which constitute primary infection stage, are released which are 2.8-5.9 µm in diameter and spindle shaped with the presence of a pair of flagella (Ayers, 1944). They grow in size after their emergence from the resting spores, they fuse together, and become amoeboid when they reach the root cells of cruciferous plants: these amoeboid structures penetrate the cell walls of root cells and form young and separate thalli inside them of varying sizes where very small thalli form sporangia and very large thalli are transformed into zoosporangia arranged in compact irregular aggregations (Ayers, 1944). The infection of root hairs is favoured by high moisture (Ayers, 1944). The zoosporangia then form clusters in the root hairs and epidermal cells and then produce 4-16 secondary zoospores which penetrate into the cortical tissues initiating the secondary infection stage in cortical cells (Kageyama & Asano, 2009). They produce secondary plasmodia inside the cortical cells and induce hypertrophy causing gall formation in the roots (Kageyama & Asano, 2009). The plasmodia are finally developed into resting spores and released in the soil (Ikegami et al., 1982).
The spores of Plasmodiophora brassicae can be found over one meter deep in the soil and hence it is very difficult to eradicate the spores of this disease. Once the pathogen has been established, management strategies aimed at lowering disease incidence and severity and preventing crop losses are limited. At present, only small disease reductions have been obtained with chemical pesticides (Howard et al., 2010). Effective management of clubroot requires the implementation of an integrated disease management approach. No single control or disease management measure effectively prevents the infection process. Here are few of the management approaches that can be applied for clubroot disease: 1. Agronomic measures: There are various agronomic practices that can be applied for the management of Plasmodiophora brassicae. This includes crop rotation, management of weeds and field sanitation, use of resistant varieties, seeding and planting time of the crops, soil health management, and use of trap crops. The clean and sanitized equipment and machinery, field sanitation, and removal of weeds from the fields can be a major step in slowing down the spread and development of the clubroot disease in the field (Javed et al., 2022). According to , when susceptible canola crops were planted in rotation with non-host crops like Barley, Pea, and Oat, the clubroot severity and resting spore concentrations in the soil were decreased and yield increment was seen as compared with that of continuous cropping of either resistant or susceptible canola. For the best management of the clubroot pathogen, a standard recommendation of more than 2 years for crop rotation is provided   found out that more the seedling age during the transplantation is, the lesser is the clubroot severity. The most susceptible seedlings were of age 1 week, the seedlings with intermediate severity had age of 2 weeks, and the lowest severity of this disease was seen in the seedlings of age 3-4 weeks. Similarly, the soil sterilization should be done prior to the seeding and transplanting of crops to reduce the clubroot infestation. Thus, factors like soil sterilization, seedling age, soil and atmospheric temperature, and use of healthy seedlings of resistant varieties of crops should be considered while planting the crops to reduce the incidence of clubroot pathogen in the fields.
Similarly, soil pH is one of the most important factors for the clubroot disease development. Acidic conditions provide best environment for the development and spread of P. brassicae, so activities to increase the pH level of the soil like adding limestone, wood or ash, or calcium cyanamide, can be done for the prevention of this disease (Fox et & Dixon, 1991). There are two types of lime amendments viz. slow acting and fact acting. Slow acting lime amendments like agricultural lime and dolomite lime should be applied at fall so as to provide enough time for them to break down and work whereas fast acting lime amendments like hydrated lime and quick lime can be applied at spring since they can work fast and increase soil pH at short time (Howard et al., 2010). However, the continuous application of lime in the field is not good, so soil testing is recommended prior to liming the field. Finally, the crops like Phacelia, black grass (Alopecurus myosuroides), field poppy (Papaver rhoeas), and field pea (Pisum sativum)can be used as bait or trap crops to ensure the germination of P. brassicae resting spores without affecting the main crop, eventually reducing the number of resting spores in the field Javed et al., 2022;Zamani-Noor et al., 2022). The root exudates of the non-host bait crops stimulates the germination of resting spores of the pathogen Friberg et al., 2005). The study conducted by (Friberg et al., 2006) found out that , leek (Allium porrum), winter rye (Secale cereale), and perennial ryegrass (Lolium perenne) significantly induced the resting spore germination of P. brassicae.

Biological Approach:
Unlike chemical method, biological method of clubroot management is environment friendly and helps not only to reduce the disease incidence, but also to maintain the fertility of the soil and a better soil environment to facilitate the growth and development of the other beneficial organisms. This method uses the locally available plant extracts, animal products, biofungicides, and micro-organisms for the disease management. Among various microorganisms, most widely used micro-organisms in clubroot management in Asia, North America, and Latin America are Trichoderma sp., Gliocladium catenulatum, Streptomyces sp., and Bacillus sp.  , the endophyte A. alternatgum suppresses clubroot when inoculated before the clubroot pathogen or at the same time as with P. brassicae; the inoculation of the endophyte after the inoculation of clubroot pathogen is not effective. Similarly, Heteroconium chaetospira is also an endophyte which has shown to reduce the clubroot disease in the field plot of the Brassica plants Narisawa et al., 1998). The bio-control agents work against the pathogens through one or more of four ways, viz., competition, antibiotic production, parasitism/predation, and induced resistance/cross protection (Arie et al., 2001). Apart from these, cabbage manure has also shown promising results in the clubroot management (Ghimire et al., 2022).

Chemical Approach:
The seeds from the infected fields contain clubroot pathogen, which can be a mode for the disease transportation to a long distance; however, if the seeds are cleaned well and treated with chemical fungicides, they can contribute to the delay or prevention of clubroot infection Rennie et al., 2011;Rod, 1992).  studied the effects of various chemical fungicidal seed treatments like Prosper™ FX (clothianidin + carbathiin + trifloxystrobin + metalaxyl), Nebijin® (flusulfamide), Vitavax® RS (carbathiin + thiram), Dynasty® 100FS (azoxystrobin), and Helix Xtra® (thiamethoxam + difenconazole + metalaxyl + fludioxonil) in canola under field conditions and greenhouse conditions in western Canada. The study concluded that the seed treatments were very effective under greenhouse condition but had no significant impact on clubroot severity in heavily infested fields. The reason might be the insufficiency of these products to eliminate large number of resting spores in the surrounding environment, and the remaining spores would still be available for the infection .
Not only the seed treatment, but the soil application of fungicides can also be done for the clubroot management Mitani et al., 2003). The cyazofamid, when applied to the field heavily infested with resting spores of P. brassicae, it was found that the root hair infection and club formation in Chinese cabbage were inhibited strongly (Mitani et al., 2003). The study suggested that the resting spore germination is directly inhibited by the fungicide cyazofamid, due to which the root hair infection and club formation are inhibited. It has also been speculated that cyazofamid inhibits the primary zoospore motility (Mitani et al., 2003). In addition, (Ghimire et al., 2022; Kowata-Dresch & May-De Mio, 2012) found Nebijin (flusulfamide) at the rate of 20 Lha -1 to be the best chemical control for clubroot of crucifers. Similarly, (Gahatraj et al., 2019) reported that the use of Terrachlor 75% WP i.e. PCNB (Penta Chloro Nitro Benzene) successfully decreased the mortality of seedlings and the severity of clubroot, and also increased the canopy cover and plant height in canola. (Buczacki & Cadd, 1976) found out that in glasshouse condition, the soil incorporation of NF 48, thiophanate methyl, and benomyl did well in the control of clubroot disease. The use of Nano Silver Hydrogen Peroxide induces Systemic Acquired Resistance (SAR) and limits the infection by the pathogen (Gahatraj et al., 2019).

CONCLUSION
Since the resting spores of Plasmodiophora brassicae persist long in soil and are mostly deep in the soil, the management of this disease is difficult and hence, it is considered one of the devastating diseases in cruciferous crops. Moreover, it can cause total crop failure. The pathogen has not been cultured in axenic media despite many efforts, and hence, infected callus culture has been used for the study of its life cycle. The resting spores germinate to produce primary zoospores which come in contact with the root hairs to cause primary infection. They reproduce in the root cells producing primary plasmodium which produces secondary zoospores and initiate the secondary infection stage in the cortical cells. This stage produces certain chemicals which induces hypertrophy and cause formation of galls in the roots. The aboveground symptoms of this disease include wilting, stunting, yellowing of leaves, and premature death of plants whereas underground symptoms include the formation of galls which block the pathway for water and nutrients from the roots to the upper parts of the plants. The aboveground symptoms appear at later stages of this disease and hence, for the identification of this disease at the earlier stages, one should consider uprooting the plants and see for the symptoms in the roots. For the management of this disease, no single method has been found highly effective. Thus, a combination of different strategies with agronomic, biological, and chemical strategies is required for its proper management.