Biological Control Of Plant Diseases Research Articles

<i>In Vitro</i> Antagonistic Activity of <i>Trichoderma harzianum</i> and <i>Bacillus amyloliquefaciens</i> against <i>Colletotrichum acutatum</i>

Biological control of plant diseases makes extensive use of the antagonists. The effectiveness of the control depends on the choice of an effective antagonist strains from criteria of implying a good knowledge of biological peculiarities of the material used. In this study, the antagonistic activities of nine isolates of Bacillus amyloliquefaciens and one isolate of Trichoderma harzianum were tested in vitro against seven strains of Colletotrichum acutatum, the causal agent of strawberry anthracnose. It was found that T. harzianum and B. amyloliquefaciens strains showed the ability to inhibit the mycelial growth of the pathogen by dual culture technique by more than 50 percent. The nonvolatile substances produced by the antagonists showed high inhibition percentages that are more than 99 percent, meanwhile their volatile compounds inhibited the mycelial growth of C. acutatum strains with low inhibition percentages starting from 30 percent except the substances produced by B. amyloliquefaciens Bc2 that showed a high inhibition percentage of more than 70 percent.

Managing the root rot disease of sunflower with endophytic fluorescent Pseudomonas associated with healthy plants

Use of pesticides is an effective way to control plant diseases but their excessive or misuse of chemical pesticides can cause environmental pollution. Whereas, biological control of plant diseases with saprophytic bacteria and fungi still giving inconsistent results, utilization of endophytic bacteria in plant disease management has been appeared as a new hope. In current study, 40 isolates of endophytic fluorescent Pseudomonas (EFP) were isolated from 20 healthy plants grown in experimental field of Karachi University and agricultural fields of Karachi and its suburb and identified according to Bergey's manual. Identification of ten potential isolates was also confirmed using genus-specific primer set i.e. PA-GS-R F 5′-CACTGGTGTT- CCTTCCTATA-3′ and PA-GS-F 5′-GACGGGTGAGTAATGCCTA-3’. Phylogenetic reconstruction associated the fluorescent Pseudomonas as a descendent of P. monteilii and P. aeruginosa. Out of 40 isolates, 10 caused growth inhibition of all 4 test root rotting fungi Macrophomina phaseolina, Rhizoctonia solani, Fusarium solani and F. oxysporum in vitro. Most of them were found to produce hydrogen cyanide, volatile antifungal metabolites, ammonia and indole acetic acid besides solubilizing phosphate. In field plot experiments five selected isolates of EFP were applied as soil drench, significantly suppressed root rotting fungi of sunflower. EFP-121 was found highly effective in improving plant height and fresh shoot weight, than other isolates and carbendazim, a fungicide. Endophytic fluorescent Pseudomonas associated with healthy plants are seems to be an effective alternate to chemical fungicides.

Compatibility of MO PLUS biofertilizer and Paenybacillus polymyxa to stimulate rice germination

The level of productivity of rice plantations can be increased through technical improvements in culture, namely spur plant growth. Increasing the germination rate is done by using growth regulators. Plants have limited synthesizing growth hormones in support of optimal growth. Therefore, it is necessary to add additional growth hormone from outside which can be given through fertilizers and symbiosis of microorganisms, including through the help of growth-regulating bacteria. MO Plus which is a combination of biological fertilizers and microorganisms Bradyrhizobium japonicum which is forming soybean root nodules to take nitrogen directly from the air and Streptomycetes as biological control of plant diseases. Efforts to merge MO Plus with P. polymyxa isolates in a consortium of biological product formulations need to be initiated to stimulate rice germination. The research stage consisted of synergistic tests by growing P. polymyxa bacteria and microbial consortium and calculating the number of bacterial colonies 48 hours in liquid media Nutrient Broth. The ability test produced IAA using L-Triptofan as a precursor, and the ability to produce GA3. The results showed that the levels of IAA and GA3 in Mo plus were 0.095 and 2.225 µg.l−1 respectively, while P. polymyxa: 0.087 and 2.251 µg.l−1. Synergism test results show that P. polymyxa and MO Plus bacteria can be grown on the same media simultaneously with the number of colonies of 2.3.108 CFU/ml. The treatment of MO Plus + Paenybacillus has the best influence on germination, wet weight, and dry weight of rice seeds.

The addition of various carbon sources on growing media to increase the siderophore level of fluorescent pseudomonad bacteria

Fluorescent pseudomonad is a member of the rhizobacterial groups which has the potential to be developed as an agent for inducing plant resistance. Several species of fluorescent pseudomonad are capable of producing siderophore. Siderophore is an organic antimicrobial compound that plays a role in biological control of plant diseases. The aim of the study was to determine the best carbon source added to the growing media to increase the levels of siderophore from the fluorescent pseudomonad PfCas3 and PfLAHp2 isolates. This research is an experimental study that use a completely randomized design with 4 treatments, which is the addition of carbon sources of glucose, fructose, glycerol on growing media, and King’s B medium (control), and 3 replications. Detection of siderophore was measured with a spectrophotometer at a 410 nm wavelength (λ). The data obtained were analyzed using ANOVA and DNMRT continued testing at a significant level of 5%. The results showed that the best carbon source added to the fluorescent pseudomonad PfCas3 isolate growing medium was glucose, with the siderophore level produced 1,574 at λ = 410 nm. Glucose added to the growing medium can also increase the levels of siderophore from the fluorescent pseudomonad PfLAHp2 isolate, which is 1,464 at λ = 410 nm.

Streptomyces griseocarneus R132 controls phytopathogens and promotes growth of pepper (Capsicum annuum)

Endophytic actinomycetes are important for the prospection of biologically active compounds and the development of biotechnological processes in agriculture for the biological control of plant diseases and phytopathogens. Eleven endophytic actinobacteria species isolated from Paullinia cupana were screened for in vitro antibiosis against five phytopathogens and control of anthracnose in pepper (Capsicum annuum) fruits. Cultures of the most active actinobacteria were fractionated with methanol and ethyl acetate, and the anthracnose control capacity of the resulting extracts was evaluated. Actinobacteria-mediated growth promotion and control of root pathogens in pepper plants were evaluated in a greenhouse. Streptomyces griseocarneus R132 (i) inhibited the growth of phytopathogens the most strongly, ranging from 57.24 ± 4.54% (Fusarium oxysporum 46.7) to 73.93 ± 3.71% (Botryosphaeria dothidea CFMAC3); (ii) markedly controlled the development of anthracnose symptoms caused by Colletotrichum gloeosporioides MPU99 in pepper fruits and plants; and (iii) promoted growth of pepper plants, increasing 42% of shoot dry mass. Actinobacteria extracts effectively controlled the anthracnose symptoms and reduced the lesion size by 30% in pepper fruits. Streptomyces griseocarneus R132 is a promising tool for the biological control of both pre- and post-harvest plant pathogens and growth promotion in pepper.

Screening microbe producing chitinase for inhibiting Ganoderma boninense

Ganoderma boninense is a serious fungal disease called basal stem rot (BSR) in oil palm plantation where the microbial ecosystem has been destroyed it could thrive and occupy the space that was created by sterilizing the soil. Infections by Ganoderma spp. begin their attack in the palm roots and gradually spread to the bole of the stem where they cause dry rot, which prevents absorption and transport of nutrients. Chitinoloytic microbes when in contact with chitin secrete an enzyme called chitinase to mainly degrade the chitin and utilize it as an energy source. The enzyme chitinase inhibits fungal growth by hydrolyzing the chitin present in the fungal cell wall resulting in inhibition of further progress of the fungi. There has been a lot of interest in this biological process because of its potential to be an agent for the biological control of plant disease and for engineering plants for resistance to phytopathogenic fungi by inducing the systemic resistance. Nine potential chitinase isolated from soil were tested for inhibit Ganoderma boninense. Based on the observations, it is the one isolate can inhibit the growth of Ganoderma boninense with activity of 0.02 U/mL.

Optimization of culture conditions for promoting heat-stable antifungal factor production level in Lysobacter enzymogenes.

Heat-stable antifungal factor (HSAF), which was first isolated from Lysobacter enzymogenes, exhibits inhibitory activities against a wide range of pathogens; however, a low level of HSAF was obtained from L. enzymogenes cultured in 0.1 × tryptic soy broth (TSB), an amount that does not satisfy HSAF application in disease control. In this study, the optimization of media components and environmental conditions were examined for improving the production of HSAF from L. enzymogenes OH11. The one factor at a time method was used to screen optimal nitrogen and carbon sources and inorganic salt. Then the orthogonal matrix method was used to determine the optimal concentration of the media components and environmental factors. The results showed that the maximum level of HSAF (23361 mAU·s) was achieved when OH11 cultured in the media of 0.7% (w/v) soybean powder, 0.5% (w/v) glucose and 0.08% CaCl2 at 200 rpm at 30°C for 60 h, which is much higher than that cultured in 0.1 × TSB. This opens up the possibility of HSAF or L. enzymogenes utilization for biological control of plant disease.

Bacillus based microbial formulations: Optimization of the production process

Bacillus sp.-based microbial formulations have found wide application in many fields: from pharmacy and medicine to environmental protection and agriculture due to the ability of this species to produce various metabolites and to form endospores. Recently, these products have gained popularity as biopesticidal and phytostimulatory agents, which are a ?green? alternative to overused agrochemicals. In order to obtain a high-quality and long-lasting product with desired characteristics, it is necessary to optimize the production process at each stage, which implies coordinating the microbial species, the type and the conditions of microbial cultivation along with formulation technologies. This paper provides a concise overview of the most important findings in this area, regarding characteristics of microbial formulations and specific criteria that need to be met when such a product is formulated. It should serve as a beginning point for everyone starting new research, not just in the field of biofertilization and biological control of plant diseases, but generally in the field of biochemical engineering.

복합기능미생물 Bacillus velezensis GH1-13을 이용한 미생물제형 개발 및 생물학적 특성

Biological control of plant diseases including bacterial and fungal pathogens has been deliberated as a viable alternative method to harmful and expensive chemical control. Bacillus velezensis GH1-13 strain was reported as a multifunctional biological agent which inhibits the plant pathogens and utilized in sustainable agriculture for improving growth of the crops. The objective of this study is to evaluate the biological potential of Bacillus velezensis GH1-13 for effective use in agriculture and industrialization. The microbial formulations of GH1-13 strain were performed into liquid, powder, granule and pellet types. Total viable cells of four microbial formulations were verified more than 107 cfu/mL(g) during 8 weeks at 40℃, and the storage stability was considered under severe condition. Plant growth promoting of four formulation types were examined to pepper and tomato plants. The powder type showed the best effect such as stem length 25.6±0.72 cm, 10 petiole and stem length 35.3±0.46 cm, 11 petiole of pepper and tomato plant, respectively. Biocontrol effect of liquid and powder types was also tested against Colletotrichum gloeosporioides causing pepper anthracnose. The resulting, 100 fold diluted liquid type exhibited control effect of 65%, and side effect on pepper and tomato plants were not detected. From the results, we suggest that microbial formulations have significant effects as biological agents such as plant growth promoting and controlling the phytopathogenic fungi. In addition, we expect that microbial agent may be useful in agriculture, although further study is needed.

The influence of carbon sources on the production of siderophores from the fluorescent pseudomonad bacteria

Abstract. Pseudomonad fluorescent is one of the rhizobacteria groups that could potentially be developed as a crop endurance inducer. Several species of fluorescent pseudomonad are able to produce siderophores. Siderophore is an antimicrobial organic compound that plays a role in biological control of plant diseases. This study aims to determine the best carbon source for the production of siderophores from the fluorescent pseudomonad isolates PfCas3 and PfLAHp2. The carbon sources are fructose, glucose, and glycerol. Detection of siderophores was measured using a spectrophotometer at a wavelength of 410 nm. The results showed that the best growth medium for producing siderophores was KB + glucose medium for both PfCas3 and PfLAHp2 isolates. The best combination was the use of PfCas3 isolate with the addition of carbon glucose source which resulted in the production of siderophores of 1.574. Keyword s : Fluorescent pseudomonad , carbon sources , siderophore

Functional analysis of polyketide synthase genes in the biocontrol fungus Clonostachys rosea

Clonostachys rosea is a mycoparasitic fungus used for biological control of plant diseases. Its genome contains 31 genes putatively encoding for polyketide synthases (PKSs), 75% of which are arranged in biosynthetic gene clusters. Gene expression analysis during C. rosea interactions with the fungal plant pathogens Botrytis cinerea and Fusarium graminearum showed common and species-specific induction of PKS genes. Our data showed a culture media dependent correlation between PKS gene expression and degree of antagonism in C. rosea. The pks22 and pks29 genes were highly induced during fungal-fungal interactions but not during pigmentation, and gene deletion studies revealed that PKS29 was required for full antagonism against B. cinerea, and for biocontrol of fusarium foot rot on barley. Metabolite analysis revealed that Δpks29 strains has a 50% reduced production (P = 0.001) of an unknown polyketide with molecular formula C15H28O3, while Δpks22 strains lost the ability to produce four previously unknown polyketides named Clonorosein A-D. Clonorosein A and B were purified, their structures determined, and showed strong antifungal activity against B. cinerea and F. graminearum. These results show that PKS22 is required for production of antifungal polyketide Clonorosein A-D, and demonstrate the role of PKS29 in antagonism and biocontrol of fungal plant diseases.

Kombinasi Biopestisida Formulasi Bacillus subtilis BNt8 dan Pestisida Nabati untuk Pengendalian Penyakit Hawar Daun Bipolaris maydis pada Jagung

Maydis leaf blight disease caused by Bipolaris maydis can attack corn plants in the vegetative and generative phase and can reduce yield up to 70%. Biological control of plant diseases with the use of biopesticides alone or in combination with other botanical materials can be efficient because it is easy to obtain, cheaper price, also environmentally friendly. The objective of the study was to obtain a combination of B. subtilis formulation with a botanical pesticide which has the potential to control the growth and development of maydis leaf blight. In addition to this, we also observed the effect of the application of the combination of botanical pesticides and B. subtilis formulation on plant growth. The study was conducted on February till June 2016 at the Plant Pathology Laboratory of ICERI with a complete randomized design and the Bajeng Experimental Farm using a randomized block design. In vitro testing, the treatment with vegetable pesticide of clove leaf extract gave better results in suppressing B. maydis. Similarly, in field testing, the application of B. subtilis formulation combined with clove leaf extract also gave a better effect in suppressing the development of maydis leaf blight disease and increasing yield. Applications of B. subtilis formulation can suppress maydis leaf blight disease by up to 13% and potentially increase yield by 26%.

Biocontrol of plant diseases is not an unsafe technology!

In their opinion paper "The unpredictable risk imposed by microbial secondary metabolites: how safe is biological control of plant diseases?" (J. Plant Dis. Prot. 124, 413-419; https://doi.org/10.1007/s41348-017-0109-5 ), H.B. Deising, I. Gase and Y. Kubo criticize the use of microbial pesticides in plant protection. They point to the ability of microorganisms to form toxic metabolites and fear severe health problems when antagonistic microorganisms are increasingly released into agro-ecosystems. In our opinion, this view fails to reflect the reality because it largely ignores the ecology of microorganisms. In this contribution, we state reasons why biocontrol of plant diseases is a safe technology.

Suppression of root-knot disease in Pogostemon cablin caused by Meloidogyne incognita in a rhizobacteria mediated activation of phenylpropanoid pathway

Biological control of plant diseases using Plant Growth Promoting Rhizobacteria (PGPR) is an emerging technique, not only because it provides control over the disease, but also acts in an eco-friendly way. In this study the isolated Pseudomonas putida strain, BG2 (KU312064.1) and Bacillus cereus BC1 (KX762284), promoted growth as well as provided resistance to Meloidogyne incognita infection. From the gene expression analysis it was found that both the strains activated phenylpropanoid pathway through – overproduction of Phenylalanine ammonia lyase (PAL) enzyme. Further, the BG2 strain also activates the chalcone synthase enzyme leading to the synthesis of flavonoids, which are known to possess nematicidal activity. We finally concluded that the BG2 and BC1 strains can be a potential biocontrol agent to deal with root-knot disease of Patchouli caused by M. incognita, and of benefit for the overall growth of the plant including increase in essential oil yield.

Potencial antifúngico de extratos brutos de Trichoderma spp.

Abstract Antibiosis is the mechanism by which certain microorganisms respond to the presence of others, secreting compounds or metabolites capable of inhibiting or impeding their development. The crude extract of Trichoderma contains a mixture of secondary compounds, which may show antibiotic effect, and has been used for the prospect of this fungus for biological control and other industrial purposes. Faced with the increasing demand of agriculture for ecologically compatible alternatives for the management of diseases, this work aimed to investigate the spectrum of action of Non-Volatile Metabolites (NVMs) of Trichoderma isolates against different plant pathogenic fungi. The antagonistic potential of NVMs was evaluated through the incorporation method of the filtered liquid extract in PDA medium. The assays showed that all the NVMs produced inhibited the fungus Sclerotinia sclerotiorum similarly. On the other hand, strains CEN1245 and CEN1274, both belonging to the species Trichoderma brevicompactum, showed broad spectrum against Sclerotium rolfsii, Colletotrichum gloesporioides, Verticillium dahliae, Fusarium oxysporum and Cylindrocladium sp. The present study describes isolates producing non-volatile metabolites with broad spectrum of antifungal action, as well as pathogen-specific. The Trichoderma spp. NVMs obtained from different soil samples cultivated with vegetables, cassava and maize were efficient in inhibiting plant pathogenic fungi belonging to other patossystems, such as forest or fruit, which could increase their potential application in biological control of plant diseases. In addition, these antagonistic fungi should be studied in greater depth for the identification of bioactive molecules of industrial interest or in commercial formulations of products for biological control of plant pathogens.

Evaluation of organic amendments from agro‐industry waste for the control of verticillium wilt of olive

Biological control of plant diseases using soil amendments such as animal manure and composted materials can minimize organic waste and has been proposed as an effective strategy in crop protection. In this study, 35 organic amendments (OAs) and 16 compost mixtures were evaluated against Verticillium dahliae by assessing both the antagonistic effect on the mycelial growth of two representative isolates of V. dahliae and the effect on the reduction of microsclerotia viability of the pathogen in naturally infested soil. Eleven OAs and five compost mixtures showed a consistent inhibition effect in in vitro sensitivity tests, with solid olive‐oil waste compost one of the most effective. Therefore, a bioassay with olive plants was conducted to evaluate the suppressive effect against V. dahliae of these selected OAs and compost mixtures. Significant reduction in the severity of the symptoms of V. dahliae indicates the potential use of grape marc compost (100% disease severity reduction) and solid olive‐oil waste, combined with other OAs. Microorganism mixtures and dairy waste OAs had a potential suppressive effect when they were combined with compost, showing a 73% and 63% disease severity reduction, respectively. A mixture of agro‐industrial waste with other biological control agents is a promising strategy against verticillium wilt of olive. To the authors' knowledge, this is the first report on the effectiveness of compost extracts (compost teas) on the inhibition of natural microsclerotia of V. dahliae, and also on verticillium wilt suppression in olive with solid olive‐oil waste.

Diversity of Trichoderma species isolated from dead branches and sapwood of Theobroma cacao trees

The diversity of Trichoderma species associated with cacao trees in Bahia state, Brazil, was investigated. A total of 100 isolates, 76 from sapwood and 24 from dead branches, were characterized by phylogenetic analysis of the internal transcribed spacer (ITS) of the ribosomal DNA and the translation elongation factor (tef1) of the RNA polymerase. Sixteen known and five putative new species were identified among the isolates used in this study. Trichoderma lentiforme was the most abundant species and exclusively found in cacao sapwood while T. parareesei was the most abundant and exclusively isolated from dead branches. The knowledge on the diversity of these fungi may contribute to their future exploitation in biotechnological applications and biological control of plant diseases.

Bacillomycin D Produced by Bacillus amyloliquefaciens Is Involved in the Antagonistic Interaction with the Plant-Pathogenic Fungus Fusarium graminearum.

Fusarium graminearum (teleomorph: Ascomycota, Hypocreales, Gibberella, Gibberella zeae) is a destructive fungal pathogen that threatens the production and quality of wheat and barley worldwide. Controlling this toxin-producing pathogen is a significant challenge. In the present study, the commercially available strain Bacillus amyloliquefaciens (Bacteria, Firmicutes, Bacillales, Bacillus) FZB42 showed strong activity against F. graminearum The lipopeptide bacillomycin D, produced by FZB42, was shown to contribute to the antifungal activity. Purified bacillomycin D showed strong activity against F. graminearum, and its 50% effective concentration was determined to be approximately 30 μg/ml. Analyses using scanning and transmission electron microscopy revealed that bacillomycin D caused morphological changes in the plasma membranes and cell walls of F. graminearum hyphae and conidia. Fluorescence microscopy combined with different dyes showed that bacillomycin D induced the accumulation of reactive oxygen species and caused cell death in F. graminearum hyphae and conidia. F. graminearum secondary metabolism also responded to bacillomycin D challenge, by increasing the production of deoxynivalenol. Biological control experiments demonstrated that bacillomycin D exerted good control of F. graminearum on corn silks, wheat seedlings, and wheat heads. In response to bacillomycin D, F. graminearum genes involved in scavenging reactive oxygen species were downregulated, whereas genes involved in the synthesis of deoxynivalenol were upregulated. Phosphorylation of MGV1 and HOG1, the mitogen-activated protein kinases of F. graminearum, was increased in response to bacillomycin D. Taken together, these findings reveal the mechanism of the antifungal action of bacillomycin D.IMPORTANCE Biological control of plant disease caused by Fusarium graminearum is desirable. Bacillus amyloliquefaciens FZB42 is a representative of the biocontrol bacterial strains. In this work, the lipopeptide bacillomycin D, produced by FZB42, showed strong fungicidal activity against F. graminearum Bacillomycin D caused morphological changes in the plasma membrane and cell wall of F. graminearum, induced accumulation of reactive oxygen species, and ultimately caused cell death in F. graminearum Interestingly, when F. graminearum was challenged with bacillomycin D, the deoxynivalenol production, gene expression, mitogen-activated protein kinase phosphorylation, and pathogenicity of F. graminearum were significantly altered. These findings clarified the mechanisms of the activity of bacillomycin D against F. graminearum and highlighted the potential of B. amyloliquefaciens FZB42 as a biocontrol agent against F. graminearum.

The unpredictable risk imposed by microbial secondary metabolites: how safe is biological control of plant diseases?

Currently, a strong public and political demand for reducing chemical pesticides exists, supporting strengthening of biological plant protection programs in agriculture. Biological plant protection strategies largely depend on introducing antagonistic microorganisms to agro-ecosystems. This approach would indeed reduce the impact of synthetic chemistries and may help avoiding development of fungicide-resistant pathogen populations, but on the other hand may strongly increase the load of microbial toxins. In crops treated with biological control agents, neither the newly forming (confronting) microbial communities nor the secondary metabolites synthesized by these are known. Many microbial secondary metabolites may not only exhibit acute toxicities significantly exceeding those of fungicides, but even act as strong carcinogens. This paper discusses the risks imposed by the introduction of biological control agents and strongly suggests including secondary metabolite gene cluster expression data into the list of information required for approving any plant protection agents and releasing them to the field. Secondary metabolism gene expression data could strongly support assessing the risk imposed by microorganisms used in disease and pest control.

Effects of biocontrol bacteria and earthworms on the severity of Alternaria brassicae disease and the growth of oilseed rape plants (Brassica napus)

Biological control of plant diseases through the addition of microbial biocontrol agents and the promotion of earthworms can be an environmentally friendly alternative to the chemical control of plant diseases. However, possible risks with biocontrol agents and their interactions with earthworms and other soil biota have not been well studied. The aim of this study was to assess whether the beneficial bacterium Bacillus amyloliquefaciens and the earthworms Aporrectodea caliginosa or Aporrectodea longa could reduce disease in oilseed rape (Brassica napus) challenged with the pathogen Alternaria brassicae. Plant growth and productivity were measured as plant survival, height, biomass, and flower development as well as disease index. A second objective was to assess whether the presence of the bacterium at high concentrations would influence the survival, growth, and reproduction of the earthworms. One outdoor and one greenhouse experiment were performed with Br. napus plants challenged with Al. brassicae inoculated to the plant leaves in the presence or absence of Bacillus amyloliquefaciens inoculated to the root environment and in the presence or absence of earthworms (Ap. caliginosa or Ap. longa) added to the soil. All treatments were replicated three times. In the outdoor experiment, inoculation with Al. brassicae reduced the growth of plants and the addition of Ap. caliginosa increased plant height. In the greenhouse experiment, pairwise comparisons of plants challenged with Al. brassicae showed that treatment with B. amyloliquefaciens led to significantly lower disease index than the treatment with Ap. caliginosa plus B. amyloliquefaciens, while other treatments had intermediate disease indices. The addition of Al. brassicae or B. amyloliquefaciens increased the survival and mass increment of Ap. caliginosa as a main effect when used separately but not when used in combination.This study did not give any clear indication of the usefulness of B. amyloliquefaciens for biocontrol of plant pathogens such as Al. brassicae when growing plants in natural soil. In addition, no significantly positive effects from the tested earthworm species were seen.