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Clubroot Resistant Brassica Seed Collection

This super saver Clubroot Resistant Brassica Seed Collection consists of 5 seed packets of our best Clubroot Resistant varieties, giving you a saving of £2! You will receive one packet each of the following varieties:

Cabbage Kilaton AGM F1 – Summer/Autumn Cutting ball-head variety- If clubroot is a problem with brassicas in your garden, this is a ‘must grow’ cabbage for Kilaton F1 is resistant to the scourge. A Dutch white type, it gives solid, high quality heads which are great for coleslaw or cooked. Heads 2.5-4kg.

Cabbage Lodero F1 – A first red clubroot resistant variety! Produces tightly packed small to medium sized heads which are full of flavour and store well. Excellent in coleslaw or steamed where it retains its colour well.

Broccoli Monclano F1 – This quality clubroot resistant variety produces firm, domed heads with fine beads. It is high yielding and has good resistance to downy mildew which also gives it good standing ability.

Brussels Sprout Crispus F1 – If clubroot is a problem in your veg plot, this early to mid-season hybrid is for you as it is highly resistant. The dark green buttons stand in good condition on the tall plants for several weeks.

Cauliflower Clapton F1 – Clubroot a problem? Not with Clapton F1, which is resistant. This late summer and autumn cropper gives heavy, dense, very white heads of superb quality and flavour. A real breakthrough.

Clubroot resistant (CR) canola varieties are key tools used to delay clubroot establishment and manage clubroot disease on the farm. However, to prevent rapid genetic shifts in clubroot populations and subsequent loss of effective resistance in CR varieties, this valuable resource must be used judiciously in an integrated management approach, which includes practicing a diverse crop rotation with at least two years between canola crops, effectively managing weeds, sanitizing equipment and minimizing soil movement.

Clubroot galls. Credit: Brittany Hennig

Resistance classifications

Clubroot resistance in a variety should be substantiated through standard testing procedures outlined in the Western Canada Canola/Rapeseed Recommending Committee (WCC/RRC) guidelines and protocols. Varieties are compared to the susceptible check variety for clubroot infection and are assigned either resistant (R), intermediate (I) or susceptible (S) ratings.

Resistant (R) Intermediate (I) Susceptible (S)
Classifcation Less than 30% infection compared to susceptible checks in disease tests. Between 30 and 50% infection compared to susceptible checks in disease tests. More than 50% infection compared to a susceptible check.
What this means (R) varieties are not immune, but highly restrict the development of clubroot symptoms in fields with low to moderate disease pressure from resting spores in the soil. This (I) rating will mostly be used for adding rating labels to the base (R) label in multiple resistance gene varieties to specify moderate resistance against certain new strains. If there is no CR label on a variety, assume it is susceptible to clubroot. An (S) label could be added to a base (R) label to specify susceptibility to certain strains that aren’t common.
What to expect Under heavy pressure in severely infested fields, an (R) variety can show significant root galling, but may develop fewer and smaller galls than a susceptible variety. Under these heavy pressure situations and frequent use of CR varieties, clubroot populations rapidly evolve to strains that overcome the resistance. Although intermediate resistance may restrict the development of clubroot symptoms for the corresponding strains, the spore concentration in the soil will be increased. An extreme buildup of spores can occur very quickly when susceptible varieties are grown in short rotation on slightly infested fields.
Management tips To delay this shift in clubroot strains and loss of CR variety efficacy, CR varieties should not be grown in short rotations in infested fields. Varieties with additional (l) labels can provide marginally better disease protection on fields with presence of new corresponding strains, but should not be grown in fields where resistance to predominant strains has been widely defeated. Susceptible varieties should not be grown in clubroot-infested fields, or those at risk of becoming infested soon.

Clubroot pathotypes

A base resistance (R) label requires that the variety is resistant to the predominant clubroot strains or pathotypes in Western Canada. Additional ratings can be appended to the base ‘R’ label to describe resistance to specific uncommon or new pathotypes.

No CR varieties, including new ones with multiple resistance genes, are resistant to all of the clubroot pathotypes detected in Western Canada to date. As clubroot populations in infested fields become more diverse over time, and more CR resistance genes are bred into canola varieties, the usefulness of rotating CR varieties with different resistance will increase. Currently there are no tests commercially available for growers to distinguish or detect new virulent strains in their infested fields.

Careful scouting to detect early infestations to alert growers and deploy resistant varieties is of utmost importance. Waiting to use ‘R’ varieties until significant infestations have developed will create high soil spore loads and increase the probability for pathogen shifts which can rapidly defeat variety resistance.

Visit clubroot.ca to learn more. Scroll through the manage clubroot section to see a list of current clubroot resistant varieties.

Best product
for Club Root

Affecting most brassica crops (cabbage, broccoli, cauliflower, etc.), club root is a serious plant disease in North American home gardens. It is caused by the soil-borne fungus Plasmodiophora brassicae which infects susceptible plants through root hairs. Diseased roots become swollen, misshapen and deformed (clubbed) often cracking and rotting. As a result, plants have difficulty absorbing water and nutrients properly.

Plants often grow poorly and wilt during the heat of the day; plants often revive during cool nights. Outer leaves may turn yellow, purple or brown. Club root will reduce yields and can cause total crop failure.

Fungal spores can be spread by wind, water and garden tools. Disease development can occur over a wide range of conditions, but is favored by excessive moisture, low soil pH and soil temperatures between 64 and 77˚F. Spores can survive in the soil for as many as 10 years.

Treatment

  1. Fungicides will NOT treat this soil-dwelling micro-organism.
  2. Choose resistant cultivars when possible.
  3. Try to prevent the occurrence of this disease by keeping a clean garden and rotating crops.
  4. Keep in mind that the disease spores can persist in the soil for up to 20 years. If club root is present you may want to solarize the soil.*
  5. Control susceptible weeds — mustard, radish, shepherd’s purse — that may be infected to reduce potential buildup of the disease.
  6. Carefully remove infected plants and sterilize garden tools (one part bleach to 4 parts water) after use.
  7. Raise your soil’s pH to a more alkaline 7.2 by mixing oyster shell or dolomite lime into your garden in the fall. Simple and affordable soil test kits are available to check pH often.

* To solarize your soil, you must leave a clear plastic tarp on the soil surface for 4-6 weeks during the hottest part of the year. Soil solarization will reduce or eliminate many soil inhabiting pests including nematodes, fungi, insects, weeds and weed seeds.

How to Manage Pests

Clubroot—Plasmodiophora brassicae

Clubroot is a problem on plants such as cabbage, broccoli, alyssum, and nasturtium, as well as many weeds in the mustard family. During initial stages of clubroot, aboveground symptoms may be absent. Foliar symptoms include stunting, yellowing, and wilting. Extensive galling, swelling, and distortion of the roots and hypocotyl are the main symptoms of the disease. Clubroot is common in soils where Brassica spp. plants have previously grown.

Identification

Clubroot may often be confused with nematode damage. Aboveground symptoms of both disorders are similar–wilting or stunting of leaves. Digging up the plants and observing the roots is the only way to distinguish the two disorders. Roots with clubroot are heavily clubbed and may appear spindle shaped. Multiple infections of the same root cause extreme swelling and distortion. Nematodes cause distinctive galls or swellings to form on the roots, but they are not clubbed or spindle shaped. In some cases, small white or brown structures (bodies or eggs of some nematode species) may be seen.

Life cycle

The fungus that causes clubroot persists in soil for many years. Infection is favored by acid soils with adequate moisture, but infections can occur above pH 7.0. In the presence of host plant roots, spores germinate and release swimming spores, called zoospores. These zoospores infect and colonize root hairs. Later, a second type of zoospore appears that can infect the main roots. Infection and colonization by this second zoospore causes the galling and clubbing of roots. Additional spores are formed inside the galled roots and are released into the soil when roots decay. The fungus is dispersed by the movement of infected plants, especially transplants, and the movement of soil.

Solutions

Clubroot is most common in acid soils. Add lime annually to affected soils below pH 7.2. Provide good drainage. Minimize the spread of the pathogen by using pathogen-free transplants. Avoid planting plants where other infested plants in the mustard family have grown, such as broccoli and cabbage. Solarization will also give control.

Club Root

Author: Howard F. Schwartz and David H. Gent

Taxonomy Kingdom: Protozoa Phylum: Plasmodiophoromycota Class: Plasmodiophoromycetes Order: Plasmodiophorales Family: Plasmodiophoraceae Genus: Plasmodiophora Species: P. brassicae Subspecies: P. brassicae Scientific Name Plasmodiophora brassicae
Woronin Common Names and Diseases club root of crucifers

Identification and Life Cycle

Club root is caused by the fungus Plasmodiophora brassicae; and can be a destructive disease of cabbage, cauliflower, broccoli, brussel sprouts, kohlrabi, Chinese cabbage, radish, turnip, and rutabaga. It may also infect many wild mustards and cultivated ornamentals such as column stock and wallflower. Infection is most severe in acid soils, and does not occur above pH 7.0. Disease develops when soilborne spores germinate and infect young roots. The fungus spreads from cell to cell in roots, and causes a rapid growth of large cells. The pathogen is disseminated among fields in infected transplants, contaminated soil on equipment, and irrigation water. Resting spores of the pathogen survive for years in soil.

Plant Response and Damage

Disease symptoms first appear as slight wilting or flagging of leaves during the warmest part of the day. The wilting becomes progressively worse, until permanent wilting occurs. Roots of infected plants are enlarged and misshaped. If infection occurs at a single site, roots are spindle-shaped, but multiple infections result in extreme swelling and distortion.

Management Approaches

No biological control strategies have been developed for club root.

Cultural Control

Plant transplants free from club root. Prevent the introduction of the pathogen into new fields by pressure-washing equipment after working in fields with the disease. Practice a three-year or longer crop rotation to non-hosts such as small grains. Avoid reuse of irrigation tail water. Club root is not a problem in alkaline soils, and liming acid soils can provide an effective control strategy. Tolerant varieties should be planted where available, but many strains of the pathogen exist that may overcome resistance in some varieties.

Chemical Control

PCNB (pentachloronitrobenzene) incorporation into soil at planting can delay root decay from secondary pathogens, but is most effective when used in combination with cultural practices such as liming.

Product List for Club Root:

Pesticide Product per Acre Application Frequency (days) Remarks
Biological Control Agent
Serenade Soil 2-6 qt Preplant application to postplant drench, 0 days PHI.
PCNB
Terraclor 15G 125 lb Preplant incorporate Apply and incorporate 4 to 6” deep in a 12 to 16” band immediately prior to planting
Terraclor 75%WP 30-40lbs Preplant incorporate Apply and incorporate 4 to 6” deep in a 12 to 16” band immediately prior to planting
Terraclor Flowable 5.6-7.5 gal Preplant incorporate Apply and incorporate 4 to 6” deep in a 12 to 16” band immediately prior to planting

The information herein is supplied with the understanding that no discrimination is intended and that listing of commercial products, necessary to this guide, implies no endorsement by the authors or the Extension Services of Nebraska, Colorado, Wyoming or Montana. Criticism of products or equipment not listed is neither implied nor intended. Due to constantly changing labels, laws and regulations, the Extension Services can assume no liability for the suggested use of chemicals contained herein. Pesticides must be applied legally complying with all label directions and precautions on the pesticide container and any supplemental labeling and rules of state and federal pesticide regulatory agencies. State rules and regulations and special pesticide use allowances may vary from state to state: contact your State Department of Agriculture for the rules, regulations and allowances applicable in your state and locality.

Growing Brassicas on a Clubroot Infected Plot

Whilst the bad news is that once you have clubroot in your soil you are stuck with it and very unlikely to ever be clubroot free, the good news is that you can continue to grow brassicas successfully on your land with some care. In fact prize-winning cauliflowers and cabbages have been grown on clubroot infected plots using the methods below.

You Can Grow Brassicas on Clubroot Infected Land

Club Root – Plasmodiophora Brassica in the soil does not mean that you can’t grow top quality cauliflowers, cabbages and other brassicas.

Reduce Clubroot in the Soil

Clubroot gets into the plant from the cysts in the soil. The less cysts in the soil, the slower the disease will develop and the better the chances of successful cropping.

You can not eliminate the disease but you can reduce the number of infectious cysts.

Keep the Plot Weed Free

Since there are some 300 species of plants including many common weeds that are members of the crucifer family and affected by clubroot, keeping the plot weed free will help avoid a reservoir of disease building up.

Practice Good Hygiene

When brassicas are harvested, remove the roots from the ground. Promptly remove any radishes that have gone over and the same with turnips that are too long in the ground and gone woody.

Don’t compost the roots or the radish & turnips. Whilst hot composting should destroy the clubroot, cold certainly won’t. Either dispose of in your household waste or burn in an incinerator.

Don’t use green manure mustard on the plot. Mustard is a member of the brassica family and will act as a reservoir for the club root.

Rotation

Whilst any sensible crop rotation will not eliminate clubroot, if you can get a 6 year rotation it will help reduce the cyst load in the soil. A three year rotation is probably the worst thing for clubroot as the cyst load may well be at peak in years 2 and 3 after growing brassicas.

Make the Soil Unattractive to Clubroot

Clubroot, being a sort of slime mould / fungus thrives best in acid wet soils. Before planting brassicas, make sure your bed is well dug over and with heavy clays add grit or sharp sand to improve drainage. Good soils with plenty of organic matter are less attractive to disease.

Increase the pH (reduce acidity) of the soil. You can take the pH up as high as 7.5 or even 8 by adding lime, preferably in the winter prior to planting out. Neutral or slightly alkaline soils deter clubroot and brassicas like a high pH anyway.

I have heard good reports from people sterilising the soil by drenching the planting area with armillatox at 100:1 dilution, 20 litres per square metre 3 weeks before planting. Jeyes fluid may also be effective. However this is not an approved use of the products in Europe.

There is another problem with soil sterilising in that it destroys the micro-ecology of the soil. All those invisible to the eye organisms that work together to help plants take up nutrients and maintain a balance.

The use of Perlka™ as a fertiliser is said to help reduce the cyst load and improve plant performance despite clubroot in the ground. Perlka contains calcium, carbon and nitrogen fused at temperatures greater than 1,000 deg C into a fertiliser, calcium cyanamide.

In the soil Perlka slowly and steadily releases its calcium and nitrogen. Calcium from Perlka raises the soil pH in a similar manner to lime (calcium carbonate) whilst the nitrogen stimulates brassica growth and vigour helping the plant resist the disease.

Additionally, Perlka™ is said to have intrinsic fertilising properties capable of encouraging soil microbes that are antagonistic to Plasmodiophora brassicae diminishing its abilities to colonise and thrive in brassica roots and simultaneously stimulating disease resistance in the brassica plant. Maximum effect is obtained by applying 7 to 10 days before sowing or transplanting.

Starting off Leafy Brassicas in Pots

This method has been proven to work and enable good quality crops to be taken – even prize winning cauliflowers.

  • Broccoli
  • Brussels Sprouts
  • Cabbages
  • Chinese Cabbage (Pak Choi)
  • Calabrese
  • Cauliflowers
  • Kale (Borecole)
  • Romanesco

Sow into a seed tray of multi-purpose compost which has had a little lime added. I put a dusting, like icing sugar on a cake, on an inch thick layer or compost and mix well.

When the seedlings have emerged and can be handled, transplant into 3″ pots of either the same compost & lime mix or a mix of that compost and John Innes #3 at 50:50

Plant the seedlings deeply so the leaves are still above the soil level but most of the stem is buried. This encourages root growth.

Once the roots start to crowd the pot you can move on to a 5″ pot of the same compost mix or for lightly infected plots, into the ground. For very infected plots, take them on to 8″ pots. When planting out, dig a hole roughly 30cm deep and in diameter. Sprinkle heavily with garden lime.

Fill the hole with the growing compost keeping a pot in the middle the same size as the brassicas are growing in. Firm down well and then remove the pot and your plant will fit perfectly into the hole. Firm again and water well.

Growing Swedes, Turnips and Kohlrabi in Clubroot Infected Plots

Fill a 12 to the seed tray module with a similar compost mix as for the leafy brassicas. Sow two or three seeds per module and thin to one on germination.

Once the roots have started to crowd the module it is time to plant out. Before planting out add extra lime (unless the pH is neutral already) to the soil and your should be OK for a crop.

Further Information on Growing Brassicas

  • 12 Tips for Success with Leafy Brassicas
  • How to Grow Great Leaf Brassicas
  • Fertiliser Requirements of Cabbages & Leafy Brassicas
  • Brassica Pests & Problems
  • About & Controlling Cabbage Root Fly – Delia radicum brassicae
  • Guide to Club Root Plasmodiophora brassicae – Control Clubroot
  • Clubroot Resistant Varieties of Brassicas
  • Growing Brassicas on a Clubroot Infected Plot

Use an integrated management strategy to ensure the long-term profitability and sustainability of clubroot-susceptible crops in the rotation. In practical terms, this means adopting sustainable rotations, judicious use of varietal resistance, diligent monitoring, surveillance and testing, combined with good biosecurity measures, and the use of wider agronomic practices, such as drainage and liming.

Identify high-risk areas

Early detection of clubroot infection is extremely important. As patches of poor growth or establishment can have multiple causes, it is important to investigate them. Where clubroot is suspected or confirmed, establish hygiene measures around the infected area immediately and adapt rotational and agronomic plans, accordingly.

Soil tests

Conduct soil tests prior to planting any susceptible crop. Prioritise high-risk areas for tests, such as wet hollows, gateways/field entrances, recently flooded fields (especially those near infected fields) and where civil infrastructure projects are planned.

Tests are based on traditional assay techniques, such as growing susceptible bait plants in suspected infected soil, or molecular diagnostics. SRUC, FERA, PGRO and Eurofins all offer testing services. Molecular test results can show the quantity of spores per gram of soil. Although suggested spore risk thresholds have been set for vegetable brassicas, no validated thresholds exist for oilseed rape.

Visual tests

If symptoms are severe enough to result in visible above-ground symptoms, NDVI (Normalized Difference Vegetation Index) techniques can be used to help detect them (see Figure 3). However, such symptoms are not always present, especially in oilseed rape, so root systems should be monitored for galls. This is best conducted over the winter period, when fields are easier to walk and monitor.

Use the field’s tramlines to establish a sampling grid. Ideally, sampling points should be no more than 50 m apart. At each sampling point, pull up and inspect 10 plants.

Clubroot maps

Use test results to create field maps of clubroot patches. Since clubroot persists for up to 20 years, knowledge of patches remains useful for several seasons.

As patches remain relatively stable within a season, targeted treatment of affected areas can be cost-effective. Targeted treatments include the use of liming, resistant varieties or fallow/grass.

Minimise contamination

Movement of infected soil or water is the main cause of clubroot introduction and spread.

On average, farm equipment transfers 250 kg of soil, most of which is deposited close to gateways and field entrances. Ensure farm staff and contractors follow good hygiene protocols and strict biosecurity procedures. Plan machinery movements to avoid travel from infested to clean fields. Restrict access to severely infested fields. Where land is rented, ensure tenants understand and manage risks in the same manner as farmer-owned land.

Where non-agricultural personnel need to access land, ensure they have sufficient biosecurity awareness, as contamination risks can be relatively high. For example, civil infrastructure projects can involve a high level of machinery movement. In such situations, additional hygiene measures may be necessary, such as hardstanding and wash-down facilities for vehicles and machinery.

Infected animal manures, composts, green mulches and straw can also introduce spores. It is important to understand the infection risk associated with these materials. Avoid the movement of feed swedes and turnips onto clean land. Risk associated with digestate use is low, particularly if it is compliant with PAS100 or PAS110 standards, but a risk remains, especially where high volumes are spread. When working with vegetable transplants, use compost with good provenance that is guaranteed clubroot-free.

As clubroot zoospores move through soil water, ensure soils are not compacted or waterlogged.

Maintain soil pH above 7

Crops grown in acidic soils are at greater risk of developing severe symptoms. Check soil pH routinely, particularly before establishing a susceptible crop. Aim to maintain the pH to above 7: even small increases (0.5–1 pH units) in pH can decrease clubroot severity. Calcium also has a direct effect on the pathogen.

Agricultural lime products, which are associated with a spike in pH and available calcium at drilling, can significantly reduce clubroot infection. High doses of lime (applied at 8 t/ha) can reduce clubroot severity by 25%. AHDB research shows that targeted treatment of the worst clubroot-affected patches can improve economic returns, compared with whole-field approaches (Table 1).

As boron also has some activity against clubroot, any deficiencies in the soil should be corrected.

Extend the rotation

In clubroot-affected land, extend the rotation to a break of at least four years between susceptible brassica crops. Clubroot resting spores can remain viable in the soil for over 15 years but have a half-life of approximately 3.5 years. Consequently, the longer the break, the greater the reduction in the number of viable spores. Thus, extending the rotation is often the most sustainable long-term management strategy.

Cover crop mixes that contain susceptible brassica hosts should be avoided on infested land. Agricultural radish is resistant to clubroot but the mechanism is the same as in oilseed rape. There is limited evidence for reduced soil inoculum after growing radish and the advice is not to grow it in severely infested fields. Spring oilseed rape is also susceptible.

Use resistant varieties judiciously

Where a susceptible crop is grown in an infected field, a resistant swede, vegetable brassica or oilseed rape variety should be selected. However, resistant varieties are not immune to attack and small galls are likely to develop. Based on a single dominant gene, the same mechanism (Mendel) is present in all resistant crop varieties. However, recent research shows there is a high pathotype diversity in the UK and resistance is not effective against all pathotypes of clubroot. Where resistance is deployed frequently in rotations or in very heavily infested soil, resistance-breaking strains are more likely to evolve and establish. This has already started to occur in some parts of the UK. As a rule of thumb, a resistant variety should have less than 30% infection compared with a susceptible variety (volunteers and off-types should not be included in the observation). Resistance breakdown is most likely to appear in patches. These patches can act as an early warning of changes in the pathogen population. Extend the rotation, where resistance breakdown is suspected.

Across Europe, resistant varieties account for about 5% of oilseed rape seed sales. The purchase of certified seed ensures that susceptible plant numbers are minimised in a resistant variety seed batch. Resistant varieties should not be home-saved for seed.

Avoid early sowing on infested sites

Infection is most likely in warm, wet soils (optimum temperature 16–25oC) when conditions enable zoospores to disperse through soil water. Disease activity reduces as soil temperatures drop below the optimum. Typically, winter-grown crops are most susceptible to infection from August to mid-September. Crops are most vulnerable at the seedling growth stage (one to two leaves unfolded). Delayed drilling minimises the potential window of infection and helps the crop to avoid clubroot.

Control weeds and volunteers early

Cruciferous weeds, such as charlock and shepherd’s-purse, are common hosts to clubroot, along with volunteer oilseed rape. Consequently, they should be managed within and between susceptible crops. Early weed control (7–14 days post emergence), by either herbicide application or shallow disking, reduces the number of resting spores in the soil.

Fungicides

Fungicide and biocontrol options are not available for clubroot control. Calcium cyanamide, which is registered for use as a fertiliser, is reported to have some incidental effect on clubroot.

Action points

  • Keep accurate crop records of clubroot occurrence, location and intensity, and where varietal resistance has been deployed in field
  • Monitor crops carefully and assess the levels of clubroot present, especially for higher-risk fields
  • Use field maps to identify hot spots to management strategies
  • At sites with higher frequencies of susceptible crops in a rotation, increase the frequency and detail of tests
  • If levels of infection start to increase, change strategy – especially, where ‘resistant’ varieties have been deployed
  • For resistant varieties, use certified seed to minimise susceptible plant numbers. Do not home-save seed of resistant varieties
  • Manage volunteers and susceptible weeds, within and between susceptible crops, as early as possible
  • Be mindful of other susceptible crop choices when planning rotations – spring rape is vulnerable and cover crop mixes often contain susceptible species
  • Make decisions based on long-term profitability and sustainability of a field, not on a single season’s predicted margin

IDENTIFY AND TREAT CLUB ROOT

By David Marks

RECOGNISING CLUB ROOT

The first signs you will have are plants that look wilted in warm weather, the leaves may have a bluish tinge to them. Typical of this disease is that the plant recover over night and may look OK in the morning only to wilt later on. As the club root progresses the leaves will yellow and the plants will slowly die.

The absolute confirmation of this disease can only be decided by pulling up plants which are suspect and looking at the root system which will be significantly swollen, often with lumps on it. The infected roots are less able to absorb water and nutrients causing the plant above ground to suffer.

Normally at this stage of a pest / disease article we write we would describe the lifecycle to help you understand what is going on and identify any weak areas. But in the case of club root there will be no indications of the problem until it is far too late. The Latin name for this disease is Plasmodiophora Brassicae.

The disease normally spreads in summer and early autumn, at temperatures below 15°C / 59°F the fungus become inactive and the disease stops developing. Wet soil also assists in the development of Club Root.

PREVENTING AND TREATING CLUB ROOT

There are no chemical treatments currently available for club root.

Club Root is a soil borne spore which is easily spread on footwear and gardening tools. It also can exist on plants and their nearby soil so be very wary of accepting small brassica plants from fellow gardeners.

Club Root has a definite preference for slightly acidic soil so applying lime, which makes the soil more alkaline, is definitely recommended – aim to raise the soil pH above 7.2. Lime can easily be bought at almost all garden centres and should be applied to the soil at the rate recommended on the packet. Doing this will significantly reduce the effect of club root if it is already in the soil.

Club root can exist in the soil for up to 15 years so crop rotation will not get rid of it but it will significantly reduce the effects. Some weeds also harbour club root so keep the soil weed free in areas which are resting from growing brassicas.

For those with club root already in the soil raising seedlings in pots for longer than normal will allow a larger root system to develop before it is planted in the ground. This can often allow crops to be raised successfully in infected soil.

There is an old wives tale that a small amount of rhubarb stalk placed in the planting hole will assist in preventing club root but tests have proven that this has no effect.

Some varieties of brassicas show resistance to club root. These include:

Further reading about Club Root is available from the following sources:

  • A very lengthy but informative document about Club Root from Kelly L. Stewart at Edinburgh University. Click here.
  • Yara Crop Nutrition
  • An article about Club Root in oil seed rape but the same principles apply to other plants. Click here.

BACK TO MAIN VEGETABLE PAGE PAGE

Clubroot Resistant Seeds

Wednesday, 16 January 2019 | SimplySeed

What is clubroot and why choose clubroot resistant seeds?

This is a question many gardeners, especially those who are new to vegetable growing, might ask and it’s a very important subject. Clubroot (Plasmodiophora brassicae) is a soil-borne fungal disease, which affects all members of the Brassica family including many related ornamental flowering type plants. Plants that become infected with clubroot will display stunted growth and in many cases a purple or yellow colouring on foliage. They will generally show signs of wilting in warm weather only to recover later in the day when the air cools.

However, below ground level, the roots of infected plants will become swollen and greatly distorted. A vast reduction in the plant’s fine root system makes it difficult, or near impossible, for the plants to absorb adequate amounts of water and nutrients, which can result in the early death of the plant. There is no effective chemical application available for clubroot and no known cure for the disease

Controlling clubroot

Effectively controlling the clubroot fungus is very difficult as it is thought the disease can remain present in the soil for up to 20 years. The roots of infected plants will become swollen and distorted, eventually turning black as they rot, discharging further spores into the soil. Affected plants will then fail to mature and subsequently die prematurely.

Clubroot is a micro-organism that can be spread by wind and water but is very often spread locally by the use of gardening tools, equipment and footwear coming into contact with infected soil. Good hygiene, therefore, is absolutely essential. The fungus thrives in wet conditions and in soils with a low pH, which means acid type soils are more prone to harbour the disease. The addition of lime to infected soil can help raise the pH, ideally to around 7.2, and may offer limited control. Rotating crops over a 3-4 year cycle may also offer some limited control. Sterilising gardening equipment including wheelbarrows might help prevent the spread of the disease to other areas of the garden or allotment.

It’s important that infected plants are not composted but instead, removed from the site completely or incinerated.

Plants affected

All brassicas are at risk of infection if clubroot is confirmed as being present in the soil. The list includes some of the most popular vegetables such as:

Cabbage

Cauliflower

Calabrese

Brussel sprouts

Swede

Turnip

Broccoli

Radish

Also, some related flowering plants such as stocks and wallflowers can be affected. In all, around 300 plants of the cruciferous family, which includes brassicas, mustard and cress can be affected by the clubroot disease.

How to avoid clubroot

The best method of control is prevention. If your garden or allotment is made up of healthy soil then rather than risk infection and having to deal with the consequences, it’s far better to take precautions. General hygiene should be the first consideration. If you have more than one vegetable plot or kitchen garden and they are in different locations then ensure all your gardening equipment is cleaned thoroughly so that fungal spores are not inadvertently transferred. This advice also applies to footwear and wheelbarrows. Crop rotation is another way to reduce the spread of fungal infections and other soil-borne diseases.

Always buy clubroot resistant seeds for all brassica crops and any brassica related plants such as wallflowers, Arabis (rock cress) and stocks. There are many types of weeds, which are also related to the mustards and form part of the brassica family. Many of these weed plants are susceptible to clubroot, so it’s important to ensure your growing areas are kept free from weeds at all times.

Recommended clubroot resistant seeds

Due to clubroot being such a difficult disease to control, in recent years there have been a number of major breakthroughs in the cultivation and production of disease-resistant brassicas and seeds. Plants grown from clubroot resistant seeds are able to grow perfectly well on infected soil but more importantly, they will help reduce the spread of the disease over time. The main selections for clubroot resistant seeds include varieties of the most popular vegetables such as Cabbage, Brussel sprouts, Calabrese (broccoli) and Cauliflower. However, a selected clubroot resistant Swede variety seed is also now available.

Some clubroot resistant brassica seeds currently available:

Brussel Sprout Crispus

Crispus is a clubroot resistant Maximus type – WOW!…..

Average Contents : 20 seeds

Only: £2.25

In stock


Cabbage Cordesa

Unique clubroot resistant savoy cabbage…..

Av. Packet Contents : 20 seeds

Type : Savoy Cabbage

Only: £1.99

Sorry, we are currently out of stock

Cabbage Kilaton

One of the long awaited varieties that is clubroot resistant…..

Packet Contents : 20 Seeds

Type: Ballhead / Round

Only: £1.99

In stock

Cabbage Lodero

Lodero is a clubroot resistant maincrop variety…..

Average Seed Contents: 20 Seeds

Type: Red Ballhead / Round

Only: £1.79

In stock

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More varieties are sure to become available in time, including radish and turnip but until then it is recommended to only use those clubroot resistant varieties where clubroot is known to be a serious problem.

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Identification and Mapping of the Clubroot Resistance Gene CRd in Chinese Cabbage (Brassica rapa ssp. pekinensis)

Introduction

Chinese cabbage (Brassica rapa ssp. pekinensis) is one of the most important leafy head vegetables cultivated in China, Korea, and Japan. Its production has been undermined by the rapid spread of clubroot disease, resulting in major economic losses. The soil-borne obligate plant pathogen Plasmodiophora brassicae Woronin causes clubroot in Brassica crops, blocking nutrient and water transport (Voorrips et al., 2003). The life cycle of P. brassicae has not been well understood until now. The P. brassicae infection starts from primary zoospores releasing and causes root hair infection and then primary zoospore or secondary zoospores induce cortical infection leading to the formation of galls on the roots (McDonald et al., 2014). Crop rotation and application of fluazinam and cyazofamid can effectively reduce the viability of resting P. brassicae spores and prevent infection (Ransom et al., 1991; Suzuki et al., 1995; Wallenhammar, 1996; Mitani et al., 2003; Townley and Fox, 2003; Miller et al., 2007; Kutcher et al., 2013; Peng et al., 2014). Although these approaches alleviate the symptoms of clubroot, they do not eradicate the disease. Breeding of clubroot-resistant (CR) cultivars is a desirable strategy for controlling clubroot owing to its advantages such as low cost and environment friendliness.

CRa, CRb, CRc, CRk, Crr1, Crr2, Crr3, Crr4, PbBa3.1, PbBa3.3, and QS_B3.1 genes have been identified in European fodder turnips (Matsumoto et al., 1998; Suwabe et al., 2003, 2006; Hirai et al., 2004; Piao et al., 2004; Sakamoto et al., 2008; Chen et al., 2013; Pang et al., 2014). Most of these genes are from different genetic resources and are associated with distinct P. brassicae pathotypes. CRa and CRb were resistant to race 2 and race 4, respectively. Crr1, Crr2, and Crr4 from Siloga were resistant to Ano-01 and Wakayama-01. CRc and CRk from Debra were resistant to isolates M85 and K04. Crr3 was detected from Milan White which was resistant to isolate Ano-01. PbBa3.1 and PbBa3.3 were detected from ECD04 conferring resistance to Pb2 and Pb7, respectively. Several CR genes were also recently mapped in B. rapa (Yu et al., 2016; Huang et al., 2017). Some of the genes and loci including CRa, CRb, and QS_B3.1, Crr3, CRk, and PbBa3.3 were clustered in a proximal region of chromosome A03 in B. rapa; whether they represent a single or multiple genes remains to be determined. CRa and Crr1a have been cloned and are known to encode Toll-interleukin-1 receptor-like domain-nucleotide binding site-leucine-rich repeat (TIR-NBS-LRR) proteins. It was reported that CRb is the same as CRa (Hatakeyama et al., 2017); however, the identities of the remaining CR genes require confirmation.

Molecular markers linked to CR loci or genes are essential for pyramiding several CR genes into one cultivar through marker-assisted selection (MAS). MAS has been successfully used for transforming CR genes into Chinese cabbage (Yoshikawa, 1981; Zhang et al., 2012). However, B. rapa, Brassica oleracea, and Brassica napus plants known to harbor genes conferring specific resistance to P. brassicae (Rocherieux et al., 2004; Werner et al., 2008; Chen et al., 2013) have all exhibited loss of resistance within a few years (Tjallingii, 1965; Kuginuki et al., 1999). Therefore, identifying novel CR genes or alleles associated with resistance to different pathotypes is essential for overcoming the challenges of co-existing pathotypes and the rapid mutation rate of P. brassicae in the field.

Next-generation sequencing (NGS)-based bulked segregant analysis (BSA) is a powerful tool for mapping disease resistance gene/genes that has been applied to Arabidopsis, rice (Oryza sativa), sorghum (Sorghum bicolor), soybean (Gycin emax), wheat (Triticum turgidum), and cotton (Gossypium; Trick et al., 2012; Yang et al., 2013; Uchida et al., 2014; Han et al., 2015; Song et al., 2017; Zhu et al., 2017). In the present study, we found the Chinese cabbage inbred line “85-74,” which exhibited resistance to the local pathogen “LAB-19” (race 4) and was distinct from CR germplasms harboring CRa, Crr1, and Crr3. We employed NGS-based BSA to identify the CR gene/genes in “85-74.” Our results can provide a basis for breeding new CR cultivars of Chinese cabbage.

Materials and Methods

Plant Materials

An analysis of CR loci-/gene-linked markers revealed that “85-74” and “CR-73” harbored the Crr3 gene, but showed distinct responses to three local pathogen isolates. We therefore crossed “85-74” and “BJN3-1” and then self-pollinated the offspring to produce an F2 population; 432 F2 individuals were used for genetic analysis and 127 were self-pollinated to generate an F3 population that was used for CR tests.

Pathogen Inoculation and CR Tests

A total of 11 field isolates were collected from infected Chinese cabbage or canola from different province of China and maintained on the roots of a susceptible Chinese cabbage “91-12” and stored in -20°C until required. These pathogens were classified according to Williams’ clubroot differential set and used to evaluate the resistance of eight Chinese cabbage germplasms; additionally, 24 seeds from each of eight CR Chinese cabbage inbred lines along with Williams’s differential set were grown for pathogen screening in the spring of 2014.

A total of 36 seeds of F1 individuals and 432 seeds of F2 individuals of “85-74” and “BJN3-1” were grown and inoculated with “LAB-19” in 2015; 36 seeds from each of 127 F3 populations were grown for the “LAB-19” inoculation test in 2016. All plants were grown in 72-well multipots and maintained in a greenhouse at 20–25°C under a 16-h photoperiod. Resting spores were prepared and inoculation was performed as previously described (Pang et al., 2014). For inoculation, 1 ml of spore suspension was applied to the bottom of the stem base of each 7-days-old seedling; disease resistance was evaluated 6 weeks later.

DNA Extraction and Pool Construction

Young leaves from eight Chinese cabbage germplasm were collected and used for characterization of known CR loci/genes. Young leaves from 127 F2 individuals were sampled and used for genome sequencing and gene mapping. The genomic DNA was extracted according to the cetyl trimethylammonium bromide method (Li et al., 2010), with minor modifications. DNA concentration was determined with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). Highly resistant and susceptible F3 families were selected and equal amounts of DNA from selected F2 individuals were mixed together to form resistant and susceptible pools (R- and S-pool, respectively) that were sequenced along with “85-74” and “BJN3-1” with the HiSeq 2500 system (Illumina, San Diego, CA, United States).

Analysis of Markers Linked to Previously Identified CR Genes

Chinese cabbage germplasms were genotyped for known CR loci/genes identification using linked markers (Saito et al., 2006; Suwabe et al., 2006; Sakamoto et al., 2008; Ueno et al., 2012; Chen et al., 2013; Hatakeyama et al., 2013; Pang et al., 2014; Zhang et al., 2014). DNA fragments were amplified by PCR and the susceptible “BJN3-1” line was used as a control. PCR products were separated on a standard agarose (2%) or polyacrylamide (6%) gel and their sizes were compared to that of known resistance genes.

Sequencing and Bioinformatics Analysis

Genome sequencing of the two parental lines and R- and S-pools and statistical analysis of bioinformatics data were carried out by Annoroad Co.1 A library of 300–500-bp insert size was constructed and paired-end sequenced on an Illumina HiSeq 2500 platform. Raw data were processed with Perl scripts to ensure data quality for subsequent analyses. The adopted filtering criteria are as follows: (1) remove the adaptor-polluted reads (reads containing more than five adapter-polluted bases were regarded as adaptor-polluted reads and would be filtered out); (2) remove the low-quality reads; reads with the number of low-quality bases (phred Quality value less than 19) accounting for more than 50% of total bases are regarded as low-quality reads; and (3) remove reads with number of N bases accounting for more than 5%. As for paired-end sequencing data, both reads would be filtered out if any read of the paired-end reads are adaptor-polluted. The obtained Clean Data after filtering will be carried out on statistics analyses on its quantity and quality, including Q30, data quantity, base content statistics, etc. Clean reads were aligned to the reference genome sequence “Chiifu-401-42” from the Ensemble Genome database2 using Burrows–Wheeler Aligner v.0.7.12 (Li and Durbin, 2009). Samtools v.1.2 (Li et al., 2009) was used to sort the reads, and duplicate reads obtained by PCR were removed using the MarkDuplicates command of Picard tools v.1.133. Reads mapped to two or more sites were filtered out. Statistical analyses were carried out using an in-house Perl script.

The Genome Analysis Toolkit (GATK; McKenna et al., 2010) HaplotypeCaller function was used for single nucleotide polymorphism (SNP) and insertion-deletion (InDel) calling. The SNPs and InDels were filtered with the GATK VariantFiltration protocol before further analysis with the following settings: QD < 2.0, MQ < 40, DP < 4, MQRankSum < -12.5. Annotation was performed using ANNOVAR (Wang et al., 2010) for all qualified variants based on the GFF file.

BSA Mapping Using Sequencing Data

To detect candidate loci associated with CR, the SNP index was calculated for all variants (Takagi et al., 2013). To reduce the impact of sequencing and alignment errors, we filtered out variations that met any of the following conditions: (1) loci in parents were heterozygous; (2) depths of variation were <10 or positions were not covered in parents or bulks; (3) variations in SNP index in both bulks were <0.3 or >0.7; or (4) variations were not on chromosomes (e.g., they were on a scaffold). All remaining variants were retained for further analysis. We slid along the genome with a 1-Mb window at a step size of 100 kb to calculate the mean SNP index, and subtracted the SNP index value of the R-pool from that of the S-pool to obtain the ΔSNP index (Takagi et al., 2013). Confidence intervals at 0.1, 0.5, and 0.01 levels were determined by computer simulation; the threshold was set at a 0.01 confidence level to identify candidate quantitative trait loci. The SNPs and InDels in the confidence region were selected and validated through Sanger sequencing.

CRd Mapping and Candidate Genes Analysis

The reference genome sequence from the Ensemble Genome database was downloaded and used for marker development in the CRd candidate region. Simple sequence repeat (SSR) markers were developed using SSR Hunter v.1.3 (Li and Wan, 2005). The two parental lines along with R- and S-pools were used to develop markers linked to the CR gene. A genetic map was constructed with the developed and previously published markers using JoinMap v.4.0 (Stam, 1993; Van Ooijen, 2006). CRd closely linked markers were validated in natural population. The candidate genes in the CRd region were compared with 244 resistance genes in B. rapa4.

Semi-Quantitative RT-PCR Analysis

Total RNA was isolated from 0 day, 7 days, 10, and 13 days after inoculation (DAI) of P. brassicae of “85-74” and “BJN3-1” root tissue using an Easy-BLUETM Total RNA Extraction Kit (Invitrogen, United States). The total RNA from each plant sample amounting to 5 μg was combined with random hexamer primers in a Super Script first-strand cDNA synthesis system according to the manufacturer’s instructions (Invitrogen, United States). Complementary DNA was diluted 10-fold, and 1 μl of the diluted cDNA was used in each 20 μl PCR mixture. Sequence information from B. rapa was used for RT-PCR primers design. Standard PCR was performed, with 5 min denaturation at 94°C followed by 25 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s. The PCR products were analyzed following electrophoresis on a 1% agarose gel.

Results

Characterization of Chinese Cabbage Germplasm

To characterize CR resources of Chinese cabbage, eight inbred lines of CR Chinese cabbage were genotyped with known CR gene linked markers and infected with 11 different local isolates of P. brassicae. “CR-77” and “CR-20” were found to carry Crr1 and CRa; “CR-75” and “CR Shinki” harbored CRa and CRb; “CR-73” harbored CRk and Crr3. “85-74” was a potential carrier of Crr3, which was linked to BRSTS61. “BJN3-1” had no CR loci, and “CR-26” carried unknown CR gene/genes (Table 1). Based on Williams’ classification system, local pathogens “AHXC-68,” “LAB-16,” “LNXM-1,” “AHHS-62,” “LAB-19,” and “LNND-2” were identified as pathotype 4; “LAB-7” and “LAB-10” were pathotype 2; “HBLC-31” and “AHHS-65” were pathotype 7; and “AHHS-65” was pathotype 11. CR inbred lines showed variable resistance depending on the presence of infection with these pathogens. The negative control “BJN3-1” was infected by all pathogens. “CR-77” and “CR-20” showed resistance to all 11 pathogens; “85-74” was susceptible to “LNND-2” and “LAB-10,” but resistant to the other pathogens tested in this study; “CR Shinki” was susceptible to “LNXM-1” and “AHHS-62,” but resistance to the other pathogens; “CR-75” and “CR-26” were susceptible to “LNXM-1” and “AHHS-62,” respectively; and “CR-73” was susceptible to “LAB-19” (Table 1). “CR-73” and “85-74” showed distinct resistance responses to “LAB-19,” “LNND-2,” and “LAB-10.” This suggests that “85-74” and “CR-73” harbor different CR genes or alleles. Based on these results, “LAB-19” was selected to phenotype F2 and F3 populations derived from the “85-74” and “BJN3-1” cross.

TABLE 1

TABLE 1. Clubroot disease resistance test of Chinese cabbage germplasms and pathotypes identification using Williams’ clubroot differential set.

Phenotype Evaluation and R- and S-Pool Construction

To investigate the inheritance of the resistance to local pathogen “LAB-19,” parental lines, F1, and 432 F2 individuals were inoculated with 1 × 107 spores⋅ml-1. The “85-74” and F1 lines were highly resistant whereas “BJN3-1” was susceptible. Among 432 F2 individuals, 321 and 106 were resistant and susceptible, respectively, and exhibited a 3:1 segregation ratio at a 0.05 level of probability (Table 2). These results indicated that CR is controlled by a single dominant gene in “85-74.” Parental lines with 127 F3 families were then inoculated with “LAB-19” in 2016. R- and S-pools were constructed by selecting 19 highly CR and 16 susceptible F2 individuals depending on the F3 family phenotype.

TABLE 2

TABLE 2. Genetic analysis of clubroot resistance in the F2 population.

Sequencing Data Analysis

TABLE 3

TABLE 3. Quality control of sequencing data.

In total, 2,941,775 SNPs and InDels were detected between “85-74” and “BJN3-1” (Figure 1). The average number of sequence variations on the 10 chromosomes was 294,177, with chromosomes A09 and A10 having the highest and lowest number of variations, respectively. Chromosome A03 had a comparatively high number and density of variations in a specific region.

FIGURE 1

FIGURE 1. Distribution of SNPs and InDels on each chromosome.

Association Analysis

To calculate SNP index, we filtered out sequence variations that met the above-described conditions. Of the 2,941,775 variations, 599,797 were to calculate SNP index (Figure 2A). According to the ΔSNP index value, a 3.94-Mb candidate region from 13.57 to 17.51 Mb was identified on chromosome A03 at a 0.01 confidence level (Figure 2B). A total of 19,664 SNPs and 4450 InDels were found in the candidate region; 1953 out of 5861 variations in exons caused changes of amino acid sequence (Supplementary Table S1). Twenty pairs of primers were designed for InDels validation (Supplementary Table S2). Seven pairs of primers were not amplified PCR production and the rest of 13 primers produced single band. The PCR products were sequenced and shown exactly same with our BSA-sequencing data (Supplementary Figure S1). The candidate genes in the CRd region were compared with 244 resistance genes in B. rapa5. Four resistance genes were identified which encode TIR-NBS-LRR protein, including Bra001160, Bra001161, Bra001162, and Bra001175 with 20, 4, 42, and 81 sequence variations, respectively, in the exons.

FIGURE 2

FIGURE 2. CRd mapping by NGS-based BSA and genetic mapping approaches. (A) Genome-wide ΔSNP index Manhattan plots and marker-trait association with 0.1, 0.05, and 0.01 confidence levels. (B) ΔSNP index Manhattan plots in candidate region; the green rectangle indicates the core region. (C) Genetic/physical map of the region harboring CRd on chromosome 3.

A core region of 577 kb was found on chromosome A03 with an extremely high average ΔSNP index of 0.9631 (Figure 2B). We designed eight SSR primer pairs within this region and screened for polymorphisms between the two parent lines. Five polymorphic markers were identified including yau301, yau389, yau376, yau106, and yau108; these were used to genotype 127 F2 individuals (Figure 2C and Supplementary Table S3). The Crr3 linked marker BRSTS61 showing polymorphism between “85-74” and “BJN3-1” was used to compare the mapping locations of CRd and Crr3.

A genetic map of the region surrounding the CRd gene was constructed based on the genotypes of seven markers. CRd was mapped to a 1 cM region with the flanking markers yau389 and yau376 (Figure 2C). Alignment of marker sequences to the reference genome sequence of B. rapa revealed a physical distance between yau389 and yau376 of about 60 kb. CRd is located upstream of Crr3 was confirmed based on the physical position of Crr3 linked markers. CRd closely linked markers yau389 and yau376 were validated in natural population and the cultivars which harbor CRd gene all showed resistant to isolate “LAB-19” (Supplementary Figure S2 and Supplementary Table S4). Total four genes Bra001160, Bra001161, Bra001162, and Bra001175 which encode TIR-NBS-LRR protein were identified in the CRd candidate region.

Total four genes Bra001160, Bra001161, Bra001162, and Bra001175 which encode TIR-NBS-LRR protein were identified in the CRd candidate region. To examine the expression characteristics of these four genes from “85-74” and “BJN3-1,” we performed RT-PCR analysis with a common primer set (18S) and resistance gene-specific primer (Supplementary Table S5). As shown in Figure 3, 18S expressed in “85-74” and “BJN3-1” from 0 DAI to 13 DAI. Bra001160, Bra001161, and Bra001175 were more highly expressed in “85-74” at 13 DAI. Bra001162 was more highly expressed in “BJN3-1” at 7 DAI, 10 DAI, and 13 DAI.

FIGURE 3

FIGURE 3. Expression levels of Bra001160, Bra001161, Bra001162, and Bra001175 genes in roots of “85-74” and “BJN3-1.”

Discussion

In this study, we used an NGS-based BSA strategy to map the novel CR gene CRd in an F2 population of B. rapa. Previously identified CR loci or genes in B. rapa have been associated with resistance to specific pathotypes of P. brassicae. For instance, the CRa gene confers resistance to P. brassicae isolate M85, which belongs to race 2 according to Williams’s classification (Williams, 1966; Ueno et al., 2012), whereas CRb (Piao et al., 2004) and Crr1 (Hatakeyama et al., 2013) confer resistance to race 4 and Ano-01 (an unknown pathotype), respectively. In the present study, “85-74” showed resistance to all pathotypes of race 2, 4, 7, and 11, except for “LAB-10” (race 2) and “LNND-2” (race 4). “CR-73” showed resistance to all tested isolates except to “LAB-19” (race 4). The variable responses to “LAB-19,” “LAB-10,” and “LNND-2” indicate that “85-74” and “CR-73” have distinct genetic backgrounds. Moreover, “CR-75” was identified carrying CRa and CRb using published CR loci-/gene linked markers and “CR Shinki” harbored CRa and CRb also. However, “CR-75” showed resistant to “AHHS-62” while “CR Shinki” not which indicates that “CR-75” is highly possible carrying other unknown CR gene/genes need to explore through genetic mapping. A given pathotype identified according to Williams’s classification system is expected to produce the same response in hosts; however, pathotype 4 had different infectivity in the Chinese cabbage germplasm inoculation tests. “LAB-7” and “LAB-10” were identified as pathotype 2; however, only the latter infected “85-74” successfully. These results indicate that the Williams’s classification system has a limited capacity for distinguishing co-existing isolates.

Clubroot disease is threating all of the brassica crops. To reduce the economic losses, CR genes have been investigated in canola (Rcr1 and Rcr4, Yu et al., 2016, 2017), cabbage (Anju1, Anju2, Anju4, and GC1, Tomita et al., 2013), and Chinese cabbage (CRa, Crr1, and CRb, Ueno et al., 2012; Hatakeyama et al., 2013; Zhang et al., 2014.) These CR genes and their closely linked markers exploration in brassica crops have been greatly improved the CR breading through MAS strategy. Breeding of CR cultivars is the most environment friendliness and economically effective strategy for controlling clubroot. Plant disease resistance genes are abundant and are clustered together in the genome (Michelmore and Meyers, 1998; Wang et al., 2011). Most of the CR loci or resistance genes reported to date in B. rapa are clustered on chromosome A03 in two specific genomic regions (CRa, CRb, and QS_B3.1 at one locus and CRk and Crr3 at another). CRd is in the same genomic region as CRk and Crr3. The CRk linked marker HC688 was not polymorphic in our population. The sequences of the Crr3 linked markers BrSTS78 and BrSTS33 were searched in the Ensemble Genome database to further distinguish between CRd and Crr3, and were found to be located at 15.091 and 15.331 Mb, respectively, on chromosome A03. Meanwhile, CRd was mapped to between yau389 (15.029 Mb) and yau376 (15.089 Mb). These results confirm that CRd is located upstream of Crr3. Moreover, “85-74” and “CR-73” (harbor Crr3 resistant gene) showed different responses to “LAB-19,” “LAB-10,” and “LNND-2.” Thus, CRd is possible to be a novel CR gene distinct from those previously identified on chromosome A03 of B. rapa.

Most of the disease resistance genes encoding NBS-LRR proteins confer pathogen race-specific resistance (Flor, 1956; Meyers et al., 2003). It was previously reported that CRa and Crr1 encode TIR-NBS-LRR protein. In the present study, Bra001162 was more highly expressed in “BJN3-1” at 7 DAI, 10 DAI, and 13 DAI indicated that Bra001162 may not associate with CR, while Bra001160, Bra001161, and Bra001175 were more highly expressed in “85-74” than “BJN3-1” at 13 DAI (Figure 3). Therefore, Bra001160, Bra001161, and Bra001175 are highly possible to be candidate genes of CRd. These results will be helpful for the CRd gene cloning and validation of transgenic lines in future study.

CRd was mapped into a 1 cM region on chromosome A03 of the B. rapa genome in a small F2 segregant population. However, it was found to be anchored to a relatively short 60 kb region based on the reference genome of B. rapa, indicating the presence of a recombination hotspot at this location. Previously studies have shown that such hotspots in Arabidopsis thaliana are accession-specific or vary depending on the cross (Drouaud et al., 2006, 2007; Kim et al., 2007; Salomé et al., 2012). It is also possible that “85-74” harbors a large insertion that is not present in the reference genome.

Conclusion

We identified the CRd gene in a CR population of B. rapa. Our findings may be useful for breeding cultivars of Chinese cabbage and other Brassica crops with broad-spectrum resistance to multiple P. brassicae pathotypes.

Author Contributions

WP analyzed the data and drafted the manuscript. PF performed the experiments and data analysis. XL and ZZ helped in the data analysis and experiments. ZP conceived the study, participated in its coordination, and helped to draft the manuscript. All authors have read and approved the final manuscript.

Funding

This study was supported by grants from the National Natural Scientific Foundation of China (Project No. 31471882), the National Key Research and Development Program of China (Grant No. 2016YFD0100202-19), and the earmarked fund for China Agriculture Research System (CARS-12).

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary Material

FIGURE S1 | Agarose gel of PCR product of InDels validation primer set.

FIGURE S2 | Validation of CRd closely linked marker (A) yau376 and (B) yau389 using natural population. Lanes 1–25 are materials that have been listed in Supplementary Table S4. Black arrow indicates the clubroot-resistant band.

TABLE S1 | Details of SNP index and annotation for 1953 SNPs and InDels that can cause changes in the amino acid sequence of the candidate region.

TABLE S2 | Primers designed for InDels validation and PCR product sequences.

TABLE S3 | Markers used for genetic map construction in this study.

TABLE S4 | Materials used for natural validation in this study.

TABLE S5 | Primer set used for semi-quantitative RT-PCR analysis.

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