1 Additional application of SO3-containing lime 6 days after inoculation.
4. Clubroot Control Using Beneficial Microorganisms
The lack of effective control measures against
P. brassicae makes it necessary to explore other, novel control options. The application of biological control measures could help to reduce soil-borne pathogens in particular. However, the complex life cycle of
P. brassicae makes it difficult to apply biological control mechanisms against this pathogen. At least three phases can be used for control: (i) germination of the resting spores and/or secondary spores, which initiate (ii) primary infection of the root hairs and secondary infection of the root cortex; (iii) antagonism/competition against the developing pathogen within the host root tissue. In addition, resistance induction in host plants and changes in microbial communities in the rhizosphere soil could be biological control options
[61].
Biocontrol agents that have been explored are bacteria or fungi including oomycetes. The mechanisms mostly are parasitism, antagonism by toxic/antibiotic secondary metabolites, and/or competition. Many studies have illustrated the biological control potential against soil fungi in
sensu stricto. This refers to the direct antagonistic or inhibitory effect on the pathogen and not to an indirect effect such as plant growth promotion effect or induction of plant resistance
[62][63].
Organisms such as, e.g.,
Trichoderma spp. and
Bacillus subtilis sensu lato, are commercially employed in many control agents against a diverse group of plant pathogens
[64][65][66][67][68]. There are numerous examples that illustrate that excellent control results can be achieved in in vitro trials
[63][69]. Whereas in field trials, those successful control results often cannot be confirmed
[70].
4.1. Antagonistic Bacteria
Bacteria of the
Bacillus subtilis species complex are well studied for their biocontrol activity against plant pathogens. These bacteria have the potential to producemany hydrolytic enzymes and diverse secondary metabolites with antimicrobial properties
[71][72]. One very well-characterized biological control agent patented strain in China is
B. subtilis XF-1. Like other
Bacillus strains, it produces fengycins, which are a group of nonribosomal lipopeptides. These metabolites have fungitoxic activities and are involved in the biocontrol effect of many
Bacillus species (reviewed by
[73][74]). Resting spores of
P. brassicae directly treated with fengycins collapsed, and the cell contents leaked out
[75]. Irrespective of this, the mode of action of fengycin was demonstrated with the
B. subtilis strain NCD-2, which showed a reducing effect on clubroot; whereas, by using fengycin, defect mutants showed no effect against the clubroot pathogen
[76]. In another experiment, Chinese cabbage seeds were soaked in a fengycin-producing
B. subtilis XF-1 bacterial suspension and the bacterial culture disease incidence was reduced by 40% and 69%, respectively
[77]. The authors demonstrated that the
B. subtilis XF-1 treatment at an early stage of seedling development had the most positive effect. In the field,
B. subtilis XF-1 reduced the disease index by about 17%
[78].
The
B. amyloliquefaciens strain QST713 (formerly B. subtilis strain) is registered for commercial uses (Serenade®) in many countries
[79]. In Canada, it was tested against
P. brassicae; however, in the field, the control success is limited
[80]. Detailed greenhouse studies showed that the biofungicide Serenade® applicated as soil drench reduced the clubroot disease incidence substantially
[81][82][83]. Recently, besides a new strain of
B. amyloliquefaciens, another member of the genus,
Bacillus,
B. velezensis, was described as a biocontrol agent against
P. brassicae [84].
Several species of the bacterial genus
Lysobacter are known for their activity against soil pathogens. These bacteria synthesize many hydrolytic enzymes and antimicrobial compounds and several commercial preparations are available against soilborne fungal pathogens
[85]. By screening bacterial strains from vegetable rhizosphere soil
[86],
Lysobacter antibioticus strains were isolated whose culture filtrates reduced clubroot severity on Chinese cabbage after application as a soil drench or seed treatment. Another
Streptomyces strain,
S. platensis 3–10, was used to optimize the culture medium and reached an inhibition of resting spore germination up to 80%
[87]. Recently, a strain of
Bacillus cereus, MZ-12, isolated from the rhizosphere soil of symptomless
B. campestris (pak choi) showed an inhibitory effect on germination of resting spores. Co-inoculation of the pak choi plants with P. brassicae spores and MZ-12 resulted in a 64% reduction in clubroot gall formation
[88].
It has been shown repeatedly that the results of in vitro and greenhouse experiments cannot be achieved with field experiments. For example, the bacterial strain
Zhihengliuella aestuarii B18 isolated from rhizosphere of
Brassica juncea showed a control efficiency of 63.4% against clubroot in greenhouse tests, whereas the control effect in the field was only 49.7%
[89].
In addition to free-living microorganisms in the rhizosphere or epiphytic-living microorganisms, endophytic-living microorganisms can also contribute to biological control. Ahmed et al.
[90] provide an overview of research carried out with endophytic bacteria and fungi as biocontrol agents. Mostly, endophytic bacteria derived from the rhizosphere enter the plant and colonize the plant tissue without any negative effect on the plant
[91]. In many cases, this form of bacterial colonization contributes to the promotion of plant growth by different mechanisms
[92]. However, the antagonistic activity by endophytic actinobacteria against clubroot has been reported by Lee et al.
[93]. They isolated 81 actinobacterial strains from the surface-sterilized root tissue of Chinese cabbage. Among them, they selected three strains that showed in vivo biocontrol activities against
P. brassicae. Two of these strains were identified as
Microbispora rosea, the third strain as
Streptomyces olivochromogenes [93]. Wang et al.
[94] tested 63
Actinobacteria strains isolated from the rhizosphere of Chinese cabbage by measuring the inhibition of the germination of
P. brassicae resting spores. This resulted in six strains that were used in greenhouse and field trials against clubroot. The strain A316 showed high control values of 73.69% in a glasshouse experiment and 65.91% in a field experiment
[94].
A recent paper by Wei et al.
[95] presents a detailed analysis of bacterial metabolites with biocontrol functions against clubroot. The work shows that co-culturing bacterial species of different genera produces more relevant metabolites than culturing bacterial species of the same genera. The results reveal that bacterial interactions between genera promote the production of biocontrol active substances.
Increasingly, microbiome studies of the rhizosphere are showing that the microbial communities are complex and highly variable
[92]. Recently, a study was presented comparing heavily
P. brassicae-contaminated soils with weakly contaminated soils. The results showed that the bacterial communities of both soil types differed significantly
[96]. Furthermore, the study showed that certain groups of bacteria were mainly found in weakly contaminated soils
[96]. Such studies suggest that individual bacterial species cannot generally act as control organisms. Instead, in each soil type, the composition of microbial communities differs and one must assume and consider, for further work, that groups of organisms act together.
4.2. Antagonistic Fungi
The soil-borne ascomycetous fungus
Phoma glomerata (current name:
Didymella glomerata) has been described as a plant pathogen
[97][98] as well as a potential biocontrol agent
[99][100]. The strain
P. glomerata no. 324 produces the secondary metabolite epoxydon. Although this substance showed very weak antifungal activity, it reduced clubroot symptoms on Chinese cabbage after spraying over the infested soil (30 mL extract solution containing 250 µg mL
−1 per 180 mL pot) completely
[101]. However, this work was conducted in the 1990s and has never been commercialized.
The fungus
Clonostachys rosea f. sp.
catenulata (syn.
Gliocladium catenulatum) is widely known as a biocontrol agent. In many countries, it is commercialized as the biofungicide Prestop® with control activity against several soil-borne plant pathogens in various crops
[102]. Neither the fungus
C. rosea nor the biofungicide Prestop® had any effect on the germination and viability of the resting spores of
P. brassicae [103]. However, soil drench treatments on B. napus seedlings 7 to 14 days after seeding resulted in a reduction in clubroot severity of about 90%. The fungus colonized the plant root system and, in this way, suppressed clubroot. In addition, it appeared to induce plant resistance since some induced plant resistance-associated genes were up-regulated
[103].
The fungal genus
Trichoderma comprises several species that are well studied as biological control agents against various plant pathogens
[104][105]. In greenhouse pot experiments, the control efficiency of
T. harzianum strain T4 against
P. brassicae in Chinese cabbage was about 79%
[106]. Another work showed the control effect of
T. harzianum strain LTR-2 in Chinese cabbage in the field. The disease incidence was lowered from 96.7% (untreated control) to 51.3% (seeds treated with spores of
T. harzianum LTR-2)
[107].
Endophytic (mutualistic) fungi grow within their host plants tissue without causing visible disease symptoms
[108]. They may have beneficial effects on the plant via plant growth promotion or by suppressing plant pathogens or pests
[109][110]. The ascomycetous soil fungus
Heteroconium chaetospira has been isolated from cabbage roots and was described as an endophytic root fungus growing throughout the cortical cells
[111][112]. In greenhouse experiments,
H. chaetospira reduced clubroot in Chinese cabbage plants by 90 to 100% after inoculation with low to moderate
P. brassicae resting spore concentrations (up to 10
5 spores per g of soil). Severely diseased plants after inoculation with 10
6 spores per g of soil could not be protected by the endophytic fungus
[113]. In field experiments, the disease reduction was lower; however, there was no reduction effect at high soil moisture
[113]. Further investigations showed that
H. chaetospira induced resistance reactions in
Brassica napus plants. The phenyalanine ammonia lyase (PAL) activity, which was increased by
H. chaetospira, and the upregulated transcript levels of several genes known to be involved in inducing plant resistance (ethylene/jasmonic acid synthesis, PR-2 protein, auxin biosynthesis) served as indicators for the resistance reaction
[114]. Another endophytic fungal genus associated with plant roots is
Acremonium. The species
A. alternatum can colonize root cells of Brassicaceae. When Arabidopsis plants were co-inoculated with
A. alternatum and
P. brassicae, the disease index of clubroot was reduced by up to 50%
[115].
5. Conclusions
Clubroot management has always been a challenge for farmers and chemical as well as classical agronomic measures have not been fully successful. In the last 10 to 20 years, a great wealth of work has been carried out on the biological control of clubroot. Some of the tested biofungicides appear promising for controlling the pathogen. However, to date, none of these approaches has become established in practice. Several studies showed that under a high disease pressure, the control activity of biocontrol agents is too weak
[80][113]. Nevertheless, it is important to pursue these efforts further. There are a number of microbial control agents against clubroot, but the knowledge of the modes of action is too low (competition, hyperparasitism, antibiosis, mixed modes with induced plant resistance?). Research into the mode of action is required to bring more agents to the approval stage. However, even more complex is the nature of soil microbial interactions and the role of microorganisms of the rhizosphere for plant health is still wildly unknown
[116]. The host plant-associated microbiomes of the rhizosphere affect the development of soil pathogens and the diseases they transmit. In the past, this interaction has often been neglected in research. Instead, the focus was on individual pathogens and their host plants in their particular environment
[117]. Recently, the number of studies on the effect of soil microbiome has increased
[118]. Raaijmakers and Mazzola
[119] discussed that the functional similarity of immune-suppressed soils across many agroecosystems suggests that it may be possible to develop disease-suppressive soil microbiomes using a universal approach. Microbiome engineering is being vigorously discussed as the biocontrol method of the future
[120]. It is becoming increasingly clear that the composition of the rhizosphere microbiome is important
[121]. It has been shown that the bacterial diversity of the seed microbiome of oilseed rape differs depending on the cultivar
[122]. Should there be less emphasis on pathogen control in the future and more focus on strengthening and compensating plants and influencing plant development by plant growth-promoting rhizobacteria?
[123]. The composition of the microbiome could play an important role here. Lebreton et al.
[124] showed that microorganism communities of healthy and clubroot-diseased plants differ considerably. Much attention should therefore be paid to comparing different rhizosphere microbiomes in order to identify important microorganisms. It is becoming possible that interactions of microbial communities could make a general contribution to the control of soil-borne plant diseases
[125].