Mode of action of microbial biological control agents against plant diseases: Relevance beyond efficacy

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.</p

Biological Control Agents for Control of Pests in Greenhouses

First we describe the different types of biocontrol used in greenhouses and present examples of each type. Next we summarize the history of greenhouse biocontrol, which started in 1926, showed a problematic period when synthetic chemical pesticides became available after 1945, and flourished again since the 1970s. After 1970, the number of natural enemies becoming available for commercial augmentative biocontrol in greenhouses grew very fast, as well as the industry producting these control agents. Biocontrol of the most important clusters of greenhouse pests is summarized, as well as the taxonomic groups of natural enemies that play a main role in greenhouses. More than 90% of natural enemy species used in greenhouses belong to the Arthropoda and less than 10%, many belonging to the Nematoda, are non-arthropods. This is followed by sections on finding and evaluation of potential biocontrol agents, and on mass production, storage, release and quality control of natural enemies. Since the 1970s, production of biocontrol agents has moved from a cottage industry to professional research and production facilities. Many efficient agents have been identified, quality control protocols, mass-production, shipment and release methods matured, and adequate guidance for farmers has been developed. Most natural enemy species (75%) are produced in low or medium numbers per week (hundreds to a hundred thousand), and are applied in situations where only low numbers are needed, such as private gardens, hospitals, banks, and shopping malls. The other 25% of the species are produced in numbers of 100,000 to up to millions per week and regularly released in many of the greenhouse crops. Microbial pesticides are predominantly used as corrective treatments in greenhouse crops where natural enemies are providing insufficient control. Europe is still the largest commercial market for arthropod greenhouse biocontrol agents, and North America is the largest market for microbial control agents. We then continue with a discussion on the pros and cons of use of polyphagous predators, and the use of semiochemicals. Finally, we summarize factors that indicate a positive future for greenhouse biocontrol, as well as developments frustrating its implementation

Biological control using invertebrates and microorganisms: plenty of new opportunities

In augmentative biological control (ABC), invertebrate and microbial organisms are seasonally released in large numbers to reduce pests. Today it is applied on more than 30 million ha worldwide. Europe is the largest commercial market for invertebrate biological control agents, while North America has the largest sales of microbials. A strong growth in use of ABC, particularly of microbial agents, is taking place in Latin America, followed by Asia. The current popularity of ABC is due to (1) its inherent positive characteristics (healthier for farm workers and persons living in farming communities, no harvesting interval or waiting period after release of agents, sustainable as there is no development of resistance against arthropod natural enemies, no phytotoxic damage to plants, better yields and a healthier product, reduced pesticide residues [well below the legal Maximum Residue Levels (MRLs)], (2) professionalism of the biological control industry (inexpensive large scale mass production, proper quality control, efficient packaging, distribution and release methods, and availability of many (>440 species) control agents for numerous pests), (3) a number of recent successes showing how biological control can save agricultural production when pesticides fail or are not available, (4) several non-governmental organizations (NGOs), consumers, and retailers demanding pesticide residues far below the legal MRLs, and (5) policy developments in several regions of the world aimed at reduction and replacement of synthetic pesticides by more sustainable methods of pest management. We are convinced, however, that ABC can be applied on a much larger area than it is today. We plead in the short term for a pragmatic form of agriculture that is adaptable, non-dogmatic and combines the sustainability gain from all types of agriculture and pest management methods. We then propose to move to “conscious agriculture”, which involves participation of all stakeholders in the production and consumer chain, and respects the environment and resource availability for future generations. Were “conscious agriculture” to be considered a serious alternative to conventional farming, ABC would face an even brighter future

Development of a multiplex Q-PCR to detect Trichoderma harzianum Rifai strain T22 in plant roots

The fungal species Trichoderma harzianum is widely used as a biological agent in crop protection. To verify the continued presence of this fungus on plant roots manually inoculated with T. harzianum strain T22, a Q-PCR was designed using specific probes for this particular strain. To develop these molecular diagnostic tools, genome mining was first carried out to retrieve putative new regions by which different strains of T. harzianum could be distinguished. Subsequently, Sanger sequencing of the L-aminoacid oxidase gene (aox1) in T. harzianum was applied to determine the mutations differing between various strains isolated from the Trichoderma collection of Koppert Biological Systems. Based on the sequence information obtained, a set of hydrolysis probes was subsequently developed which discriminated T. harzianum T22 strains varying in only a single nucleotide. Probes designed for two strains uniquely recognized the respective strains in Q-PCR with a detection limit of 12,5 ng DNA. Titration assays in which T. harzianum DNA from distinct strains was varied further underscored the specificity of the probes. Lastly, fungal DNA extracted from roots of greenhouse cultured tomato plants was analyzed using the probe-based assay. DNA from T. harzianum strain T22 could readily be identified on roots of greenhouse reared tomato plants inoculated with varying concentrations up to one week after treatment with a detection limit of 3e6 colony forming units of T. harzianum T22. We conclude that the Q-PCR method is a reliable and robust method for assessing the presence and quantity of T. harzianum strain T22 in manually inoculated plant material. Our method provides scope for the development of DNA based strain specific identification of additional strains of Trichoderma and other fungal biological control agents