Mass spectrometry as a tool for the selective profiling of destruxins; their first identification in Lecanicillium longisporum.

Mass spectrometry was applied to the identification of the destruxins (dtxs), cyclic peptides that are commonly produced by the fungal insect-pathogen, Metarhizium anisopliae. The aim of the study was to optimise a methodology in order to firstly determine whether these compounds were present in other species and to determine the effect of differing growth conditions upon the dtx content detected. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF-MS) was initially used to analyse the dtxs, but limitations were indicated. Nano-scale high-performance liquid chromatography/electrospray ionisation mass spectrometry (HPLC/ESI-MS) and automated 'data-dependent' tandem mass spectrometric (MS/MS) analysis were also applied, utilising characteristic neutral losses during fragmentation to confirm the presence of the dtxs. This latter approach distinguished the dtx E and B isoforms by retention time and diagnostic neutral losses during fragmentation allowing extraction of the destruxin data from a complex dataset. This process revealed the presence of a number of dtxs in the fungal species Lecanicillium longisporum, a species previously not known to produce dtxs, and dtx production in this species was shown to be significantly higher in aerated cultures compared with still cultures.

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.

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 12,5ng 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.