Evaluating the synergistic effects of biochar and biological control agents in sustainable agriculture

Background:Septoria tritici blotch (STB) caused by fungus Zymoseptoria tritici, is one of the important wheat (Triticum aestivum L.) diseases difficult to control because of the lack of wheat resistant cultivars. The use of biological control agents is one possible way for triggering host plant resistance to biotic and abiotic stressesObjective: In this study, we examined the ability of Serendipita indica and Pseudomonas protegens CHA0-mCherry in inducing the local wheat cultivar Tajan resistance to STB.Materials and Methods: The interaction between biological control agents and the roots of wheat was evaluated. The experiment was conducted in a completely randomized design by three replicates. Spore suspension was supplied at concentrations of 107 and 109 for S. indica and bacteria isolate (CHA0-mCherry) respectively. Five treatments were applied including S. indica, CHA0-mCherry, S. indica and CHA0-mCherry co-inoculation, positive and negative control. Twenty-one days after inoculation, the interaction between biological agents and plant roots were evaluated through morphological traits and qPCR. The plant resistance, disease severity, and the correlation between resistance and disease severity were assessed. Pycnidial variation and agronomic traits were also evaluated.Results: Twenty-one days after inoculation, both biological agents clearly colonized all treated roots of all treatments except in control plants as demonstrated by qPCR analysis. Chlamydospores were observed in the S. indica-treated hosts with the CHA0-mCherry colonizing assessment showing 5×109 CFU g-1 in the root. The asexual phase of the fungal pathogen, pycnidial diameter, was reduced in S. indica treated plants more considerably than in the other treatments. There was a positive correlation between resistance and disease severity mean when calculated by Pearson’s correlation. There was a significant difference between the root length, fresh, and dry weight of root. Spore density was inversely correlated to resistance and disease severity, when compared with control, with CHA0-mCherry being the most effective in reducing the spore density. S. indica was the most effective in promoting root growth and stem biomass, when compared with control.Conclusions:Serendipita indica and Pseudomonas protegens CHA0-mCherry colonies showed a potential biological control activity and efficiently enhanced the plant resistance to Z. tritici in the treated wheat roots. The microbial biological control agents are very practical in crop protection against plant disease and can be very useful in sustainable agriculture.Abbreviations: PLSN: percentage of leave surface necrosis, DPI: day past inoculation, PLACL: percentage of leaf area covered by lesions, PPMLA: pycnidia per millimeter in leaf area.

The characterization of microbial biological control agents (MBCAs) is crucial to improve their efficacy and consistency as biopesticides. Powerful approaches to characterize MBCA’s modes of action are provided by modern molecular technologies. This paper reviews improvements achieved in this subject by three “omics” approaches: namely the genomic, the transcriptomic and the proteomic approaches. The paper discusses the advantages and drawbacks of new molecular techniques and ‘discovery driven’ approaches to the study of the biocontrol properties against plant pathogens. Omics technologies are capable of: (i) identifying the genome, transcriptome or proteome features of an MBCA strain, (ii) comparing properties of strains/mutants with different biocontrol efficacy, (iii) identifying and characterizing genes, mRNAs and proteins involved in MBCA modes of action, and (iv) simultaneously studying the transcriptome or proteome of the plant host, the plant pathogen and the MBCAs in relation to their bi- or tri-trophic interactions.

The Nagoya Protocol actions the third objective of the Convention on Biological Diversity and provides a framework to effectively implement the fair and equitable sharing of benefits arising out of the use of genetic resources. This includes microorganisms used as biological control agents. Thus biological control practitioners must comply with access and benefit-sharing regulations that are implemented by countries providing microbial biological control agents. A review of best practices and guidance for the use and exchange of microorganisms used for biological control has been prepared by the IOBC Global Commission on Biological Control and Access and Benefit-Sharing to demonstrate commitment to comply with access and benefit-sharing requirements, and to reassure the international community that biological control is a very successful and environmentally safe pest management strategy that uses biological resources responsibly and sustainably. We propose that best practices include the following elements: collaboration to facilitate information exchange about the availability of microbial biological control agents and where they may be sourced; freely sharing available knowledge in databases about successes and failures; collaborative research with provider countries to develop capacity; and production technology transfer to provide economic opportunities. We recommend the use of model concept agreements for accessing microorganisms for scientific research and non-commercial release into nature where access and benefit-sharing regulations exist and where regulations are not restrictive or do not exist. We also recommend a model agreement for deposition of microbial biological control agents into culture collections.

Microbial interactions in agricultural ecosystems are chemically dynamic, with significant implications for ecological balance and crop protection. As synthetic fungicides face increasing regulatory and resistance challenges, applying microbial biological control agents (MBCAs) presents a potential alternative crop-protection strategy. However, the chemical and toxicological consequences of such applications remain poorly understood. Using the model ascomycete Aspergillus nidulans, the response to confrontations with twelve microbial partners, including bacterial and fungal MBCAs, plant pathogens, and phylloplane isolates was studied. Dual-culture assays revealed distinct interaction patterns, and transcriptome profiling showed confrontation-specific activation of secondary metabolite biosynthetic gene clusters (SMBGCs), with up to 50 SMBGCs differentially expressed in A. nidulans. Complementary untargeted LC-MS/MS identified hundreds of unique secondary metabolites (SMs), including compounds structurally resembling synthetic fungicides, including azoles and piperidines. Notably, cytotoxicity assays using human HEK-293 and HCT-116 cell lines revealed that SM mixtures from confrontations of A. nidulans with Bacillus subtilis or Trichothecium roseum exhibited significant toxicity, with IC₅₀ values as low as 52 µg/mL. These findings demonstrate that microbial confrontations can trigger the production of diverse and specific sets of SMs, including compounds representing potential human health risks. This study underscores the need for confrontation-informed toxicological assessments in MBCA regulation and highlights the importance of developing safer biocontrol strategies in agriculture.IMPORTANCEThis study shows fundamental changes at the transcriptome and metabolome level in the ubiquitous fungus Aspergillus nidulans confronting various microorganisms, including microbial biological control agents. Strong modulations of transcript abundances of genes belonging to secondary metabolism gene clusters correlated with the formation of a vast array of novel secondary metabolites. Compounds formed in some confrontations were toxic to human cells, questioning the consumer safety of applying microbial biological control agents.

There is considerable interest in the exploitation of microbial biological control agents (MBCAs) for the control of crop pests, weeds and diseases. MBCAs can be used where chemical pesticides are banned or being phased out or where pests have developed resistance to standard chemicals. The use of MBCAs can play an important role in crop protection, as a key element in integrated pest management (IPM) programmes. However, despite considerable research efforts on the development of new biological control agents the number of such products on the market in the European Union (EU) is still extremely low compared with the USA or Canada. In areas that previously constrained the commercialization of MBCAs, discovery, fermentation, formulation and application, significant progress has been made. The low number of products is mainly due to the slow registration process. In the EU, MBCAs are regulated by and follow Directive 91/414/EEC for placing plant protection products in the market. Once an active ingredient is listed in Annex I, national registrations for the formulated product have to follow. This time consuming and expensive process has forced most companies to suspend their efforts in research and development. Initiatives by stakeholders from industry, science, regulatory authorities, policy and environment are underway to accelerate market introduction of MBCAs.

Fungal entomopathogens are widespread in nature and contribute to the natural regulation of insects. They can be exploited for pest management as biological control agents of pests in attempts to improve the sustainability of crop protection. Four types of biological control are recognized: classical, inoculation, inundation, and conservation biological control. Classical biological control is the intentional introduction and permanent establishment of an exotic biological agent for long-term pest management. Inoculation biological control is the intentional release of a living organism as a biological control agent with the expectation that it will multiply and control the pest for an extended period, but not permanently. Inundation biological control is the release of large numbers of mass-produced biological control agents to reduce a pest population without necessarily achieving continuing impact or establishment. Conservation biological control is a modification of the environment or existing practices to protect and enhance specific natural enemies or other organisms to reduce the effect of pests. The traditional and the most popular approach in biological control with entomopathogenic fungi has been to apply the fungal material to the cropping system (as biopesticide), using an inundation biological control strategy. The term biopesticide is used for microbial biological pest control agents that are applied in a similar manner to chemical pesticides. The use of biopesticides can substitute for some (but not all) chemicals and provide environmentally safe and sustainable control of pests but EU legislation and prohibitive registration costs are discouraging the development and commercialization of many promising new products.

Microbial biological control agents (MBCAs) are applied to crops for biological control of plant pathogens where they act via a range of modes of action. Some MBCAs interact with plants by inducing resistance or priming plants without any direct interaction with the targeted pathogen. Other MBCAs act via nutrient competition or other mechanisms modulating the growth conditions for the pathogen. Antagonists acting through hyperparasitism and antibiosis are directly interfering with the pathogen. Such interactions are highly regulated cascades of metabolic events, often combining different modes of action. Compounds involved such as signaling compounds, enzymes and other interfering metabolites are produced in situ at low concentrations during interaction. The potential of microorganisms to produce such a compound in vitro does not necessarily correlate with their in situ antagonism. Understanding the mode of action of MBCAs is essential to achieve optimum disease control. Also understanding the mode of action is important to be able to characterize possible risks for humans or the environment and risks for resistance development against the MBCA. Preferences for certain modes of action for an envisaged application of a MBCA also have impact on the screening methods used to select new microbials. Screening of MBCAs in bioassays on plants or plant tissues has the advantage that MBCAs with multiple modes of action and their combinations potentially can be detected whereas simplified assays on nutrient media strongly bias the selection toward in vitro production of antimicrobial metabolites which may not be responsible for in situ antagonism. Risks assessments for MBCAs are relevant if they contain antimicrobial metabolites at effective concentration in the product. However, in most cases antimicrobial metabolites are produced by antagonists directly on the spot where the targeted organism is harmful. Such ubiquitous metabolites involved in natural, complex, highly regulated interactions between microbial cells and/or plants are not relevant for risk assessments. Currently, risks of microbial metabolites involved in antagonistic modes of action are often assessed similar to assessments of single molecule fungicides. The nature of the mode of action of antagonists requires a rethinking of data requirements for the registration of MBCAs.

Plant diseases and pests are risk factors that threaten global food security. Excessive chemical pesticide applications are commonly used to reduce the effects of plant diseases caused by bacterial and fungal pathogens. A major concern, as we strive toward more sustainable agriculture, is to increase crop yields for the increasing population. Microbial biological control agents (MBCAs) have proved their efficacy to be a green strategy to manage plant diseases, stimulate plant growth and performance, and increase yield. Besides their role in growth enhancement, plant growth-promoting rhizobacteria/fungi (PGPR/PGPF) could suppress plant diseases by producing inhibitory chemicals and inducing immune responses in plants against phytopathogens. As biofertilizers and biopesticides, PGPR and PGPF are considered as feasible, attractive economic approach for sustainable agriculture; thus, resulting in a “win-win” situation. Several PGPR and PGPF strains have been identified as effective BCAs under environmentally controlled conditions. In general, any MBCA must overcome certain challenges before it can be registered or widely utilized to control diseases/pests. Successful MBCAs offer a practical solution to improve greenhouse crop performance with reduced fertilizer inputs and chemical pesticide applications. This current review aims to fill the gap in the current knowledge of plant growth-promoting microorganisms (PGPM), provide attention about the scientific basis for policy development, and recommend further research related to the applications of PGPM used for commercial purposes.

Tea is one of the most widely consumed beverages around the world. Larvae of the moth, Ectropis obliqua Prout (Geometridae, Lepidoptera), are one of the most destructive insect pests of tea in China. E. obliqua is a polyphagus insect that is of increasing concern due to the development of populations resistant to certain chemical insecticides. Microbial biological control agents offer an environmentally friendly and effective means for insect control that can be compatible with "green" and organic farming practices. To identify novel E. obliqua biological control agents, soil and inset cadaver samples were collected from tea growing regions in the Fujian province, China. Isolates were analyzed morphologically and via molecular characterization to identity them at the species level. Laboratory and greenhouse insect bioassays were used to determine the effectiveness of the isolates for E. obliqua control. Eleven isolates corresponding to ten different species of Metarhizium were identified according to morphological and molecular analyses from soil and/or insect cadavers found on tea plants and/or in the surrounding soil sampled from eight different regions within the Fujian province, China. Four species of Metarhizium including M. clavatum, M. indigoticum, M. pemphigi, and M. phasmatodeae were documented for the first time in China, and the other species were identified as M. anisopliae, M. brunneum, M. lepidiotae, M. majus, M. pinghaense, and M. robertsii. Insect bioassays of the eleven isolates of Metarhizium revealed significant variation in the efficacy of each isolate to infect and kill E. obliqua. Metarhizium pingshaense (MaFZ-13) showed the highest virulence reaching a host target mortality rate of 93% in laboratory bioassays. The median lethal concentration (LC50) and median lethal time (LT50) values of M. pingshaense MaFZ-13 were 9.6 × 104 conidia/mL and 4.8 days, respectively. Greenhouse experiments and a time-dose-mortality (TDM) models were used to further evaluate and confirm the fungal pathogenic potential of M. pingshaense MaFZ-13 against E. obliqua larvae. Isolation of indigenous microbial biological control agents targeting specific pests is an effective approach for collecting resources that can be exploited for pest control with lowered obstacles to approval and commercialization. Our data show the presence of four different previously unreported Metarhizium species in China. Bioassays of the eleven different Metarhizium strains isolated revealed that each could infect and kill E. obliqua to different degrees with the newly isolated M. pingshaense MaFZ-13 strain representing a particularly highly virulent isolate potentially applicable for the control of E. obliqua larvae.

In recent years, as a result of the introduction of the European Commission’s recommendations on limiting the use of chemical plant protection products in favor of non-chemical ones, an increase in the available range of microbial biological control agents has also been observed in field crops protection. 8 bioinsecticides and 12 biofungicides have already been registered for these crops in Poland. Strains of the entomopathogenic bacteria Bacillus thuringiensis and one strain of the entomopathogenic fungus Beauveria bassiana are mainly used to control plant pests. Strains of the antagonistic fungi Trichoderma harzianum and Trichoderma asperellum, the hyperparasitic fungus Coniothyrium minitans and the fungus-like organism Pythium oligandrum are used to control plant disease. The bacterium Pseu­domonas sp. is also used, and the bacterium Bacillus amyloliquefaciens has been registered for dressing. Intensive scientific research is currently underway to improve the effectiveness of biological control agents. Innovative formulations are being created to increase their efficacy and extend the storage period. The future will also include plant protection programs with the combined use of beneficial mi­croorganisms and macroorganisms. This publication presents currently available microbiological plant protection products used in field plant protection, analyzes current and future research on their use and improvement of formulations.

Microbial biological control agents (MBCAs) against pests and diseases in crops are regarded as sustainable tools in integrated pest management. In the European Union, biological control should be preferred to conventional chemical methods if they provide satisfactory pest reduction. There is no reason to believe that all forms of biocontrol are intrinsically safe. Therefore requirements for registration to assure safety are needed. In the current registration procedure in the European Union, MBCAs are primarily treated as potentially risky organisms that not only produce toxic substances but are also dangerous because they can multiply, spread and genetically adapt. These characteristics give rise to a concern that released MBCAs will spread and become dominant in the environment, resulting in negative effects on other organisms in the natural environment or even humans. These assumption led to extensive data requirements that are a time consuming and costly hurdle for bringing MBCAs onto the market. This paper focuses on the relevance and irrelevance of the data requirements for environmental fate and persistence of MBCAs. MBCAs are naturally occurring living organisms that are numerically enhanced to reduce specific plant pathogens or pests. In contrast to chemicals, direct toxicity is not the main mode of action, but rather a variety of mechanisms is involved, including competition, parasitism and activity of secondary metabolites. Their effects and residues cannot be evaluated as done for chemical substances and their breakdown products. Populations of introduced microorganisms always decline due to the natural biological buffering of the environment to levels that are within common fluctuations and ranges without strongly affecting microbial communities. However, the currently used concept of a natural background level as a reference to which densities of MBCAs should decline is of limited value, in particular when endpoints cannot be defined from ecological theory or risk criteria. In conclusion we state that data requirements for persistence could be more freely interpreted in all cases where there is no a priori reason to assume that organisms will not be buffered by the agroecosystem. Since information is only needed ‘when relevant’, the European Union guidelines leave space for such a proportional interpretation of the data requirements on environmental fate.

The soil borne pathogen Rhizoctonia solani AG8 causes Rhizoctonia bare patch disease, a major constraint on global wheat production, particularly in no‐ or minimal ‐till systems. Current control strategies such as crop rotation, chemical fungicides, and tillage provide only partial protection, while fungicides can accelerate resistance development and negatively impact crop, soil, and human health. Microbial biological control agents (BCAs) and microbial synthetic communities (SynComs, combining two or more BCAs) represent a sustainable alternative for managing Rhizoctonia bare patch. However, inconsistent efficacy and limited compatibility with fungicides remain key challenges to adoption. Here, we demonstrated that individual BCAs and a SynCom (comprising Diaporthe sp . and Pseudomonas sp. KAR75) reduced disease incidence at the seedling stage by 51%–71% compared with fungicide treatment. When combined with fungicide, disease incidence was further reduced by 65%–100%. The BCA treatments also improved grain yield by 9%–12% compared with fungicide treatment and by 11%–15% compared to no‐pathogen control, demonstrating their ability to promote wheat growth in the absence of disease. Grain yield increased most with the fungal‐bacterial SynCom, particularly when applied together with fungicide. These results demonstrated that integrating BCAs with chemical fungicides has strong potential to improve control of soil‐borne diseases in grain crops.

Developing sustainable agriculture by identifying non-chemical alternative Plant Protection Products (PPP) is a cornerstone in achieving long-sought environmental friendliness. Despite significant legislative and political efforts to promote biocontrol solutions and Integrated Pest Management (IPM), the literature points out the disadvantages posed by European Union’s (EU) two-tier system for Microbial Biological Control Agents (MBCA) approval and subsequent Microbial Biological Control Products (MBCP) authorization by each EU Member State (MS). Despite the disadvantages, in a recent article, we showed that the EU had outcompeted the US and other countries in approved MBCA in the last decades; however, MBCP approval at the national level lags. Achieving the EU Green Deal’s aim set out in the ‘Farm to Fork Strategy’ to reduce the use and risk of pesticides by 50% by 2030 is difficult without developing viable alternatives. Why do we not have higher MBCP availability and usage in the EU? Is it the current legislation, its poor application, or some other factors? The current legislative framework stimulated MBCA approval. Thus, we compare MBCA approval and MBCP authorization procedure to evaluate if MBCP authorization is more difficult and thus causes a bottleneck. We find that requirements for MBCP authorization are unnecessarily more complex. We recommend simplifying the MBCP dossier requirements and making them as similar to MBCA as possible to accelerate the MBCP authorization in more EU MS to increase their availability and integration in agronomic crops’ pest management plans.

Endophytic fungi are used as the most common microbial biological control agents (MBCAs) against phytopathogens and are ubiquitous in all plant parts. Most of the fungal species have roles against a variety of plant pathogens. Fungal endophytes provide different services to be used as pathogen control agents, using an important aspect in the form of enhanced plant growth and induced systemic resistance, produce a variety of antifungal secondary metabolites (lipopeptides, antibiotics and enzymes) through colonization, and compete with other pathogenic microorganisms for growth factors (space and nutrients). The purpose of this review is to highlight the biological control potential of fungal species with antifungal properties against different fungal plant pathogens. We focused on the introduction, biology, isolation, identification of endophytic fungi, and their antifungal activity against fungal plant pathogens. The endosymbionts have developed specific genes that exhibited endophytic behavior and demonstrated defensive responses against pathogens such as antibiosis, parasitism, lytic enzyme and competition, siderophore production, and indirect responses by induced systemic resistance (ISR) in the host plant. Finally, different microscopic detection techniques to study microbial interactions (endophytic and pathogenic fungal interactions) in host plants are briefly discussed.

Plant pathogenic bacteria are a substantial and long-term danger to food supply and environmental stability across the planet. With the increase in agricultural productivity in the last few decades, farmers are getting reliant on agrochemicals as a reasonably consistent means of crop protection. But increased use of chemical inputs has various negative consequences, including disease resistance to the agents used and non-target environmental repercussions. Hence biological control is being explored as an alternative or supplement to chemical control in agriculture. This study reviews the interactions taking place between a given microorganism (fungi, bacteria, viruses) and a plant. The relation between the two organisms in the discussion can be encompassed in an elaborate manner based on the mutual gains and losses incurred by both the organisms. Microbes that interact with plants, can be manipulated and used as Biological Control Agents (BCA). BCAs are potentially seen as the alternative to using harmful chemicals in the name of fertilizers and weedicides. This study illustrates the various possible interactions between plant and other species, with specific emphasis on bacterial biocontrol and tries to answer the question: How can these interactions be articulated against a third, pathogenic and harmful species?