Several members of the AEFB have gained prominence as potent BCAs due to their activities within the phytosphere, which have resulted in a number of strains being commercialised as biopesticides and biofertilisers (Jacobsen et al., 2004; McSpadden Gardener, 2004). Many of these bacteria produce a range of antimicrobial compounds active against fungal, oomycete, and bacterial plant pathogens (Velho et al., 2011; Govindasamy et al., 2010; Nagórksa et al., 2007; Pryor et al., 2007;
Emmert and Handelsman, 1999; Bélanger et al., 1998). In addition, some plant-associated AEFB species are able to contribute to plant growth promotion through various mechanisms, such as:
nutrient solubilisation, plant growth hormone production, and the stimulation of host plant resistance mechanisms (Heydari and Pessarakli, 2010; Choudhary et al., 2009; Bargabus et al., 2004;
Kloepper et al., 2004; McSpadden Gardener, 2004).
Much of the focus on AEFB as potential biocontrol agents has fallen on their activity within the rhizosphere. However, members of this grouping are also common residents of the phyllosphere, with some having been successfully applied as foliar disease antagonists (Collins et al., 2003;
Bargabus et al., 2002; Nair et al., 2002). A number of Bacillus spp. are able to establish in this habitat, and their activities within the phyllosphere have enabled their use as biocontrol agents against a range of foliar diseases, including: powdery mildew of cucurbits, Cercospora leaf spot on sugar beet, and Colletotrichum dematium on mulberry (Romero et al., 2004; Collins and Jacobsen, 2003; Yoshida et al., 2001; Bettiol, 1997). The successful colonisation of the leaf surface by introduced microbes is impacted by the manner in which biocontrol agents are applied as well as by the prevailing phyllosphere conditions (Lindow, 2006; Andrews, 1992; Andrews, 1990; Knudsen and Spurr, 1988).
Screening of candidate BCAs requires evaluation of a large pool of isolates to minimise the risk of excluding promising candidates. Dereplication steps are often included after initial isolate culture as
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a means of streamlining candidate numbers and selecting representative isolates from groupings for further study (Ghyselinck et al., 2011). Dereplication allows taxonomic-level differentiation and subsequent grouping of isolates with the aim of minimising time and resource wastage and unnecessary downstream analyses (Ghyselinck et al., 2011). Phenotypic characterisation has, until recently, been the principal means by which bacteria were identified and their species-level diversity assessed. However, this approach does not readily distinguish between closely-related organisms, and thus is unsuitable for differentiation of closely-related isolates, and cannot resolve strain-level variations (van Belkum, 1994). Hence, molecular approaches have largely superseded phenotypic characterisation methods (Li et al., 2009). Since dereplication is achieved by grouping isolates at a taxonomic level, the accessibility and resolution of genotyping methods greatly increases the ability to accurately differentiate and group bacterial isolates. There is a wide range of high-throughput PCR-based methods suitable for dereplication purposes, including commonly-used DNA fingerprinting approaches (Ghyselinck et al., 2011).
DNA fingerprinting is often employed as a means of assessing strain diversity amongst a set of bacterial isolates (Ghyselinck et al., 2011; van Belkum, 1994). This approach exploits genetic polymorphisms to differentiate between microorganisms based on differences in banding patterns generated by PCR amplicons after gel electrophoresis (Daffonchio et al., 2003; Shaver et al., 2001;
Tyler et al., 1997; van Belkum, 1994). Isolates displaying the same fingerprint can generally be assumed to belong to the same species or strain (Logan et al., 2009). Randomly amplified polymorphic DNA PCR (RAPD-PCR) and intergenic transcribed spacer region PCR (ITS-PCR) fingerprinting methods were selected for use in the present study. RAPD-PCR applies a single short primer (~ 10 bp) that anneals to compatible sites throughout the genome, which results in a series of variably-sized fragments in the PCR product (Li et al., 2009; Olive and Bean, 1999). In contrast, ITS- PCR targets polymorphic differences within the intergenic transcribed spacer (ITS) region located between the 16S–23S rRNA subunit operons, which are under less conservation pressure than the adjacent rRNA genes (Li et al., 2009; Daffonchio et al., 2003; Shaver et al., 2001; Nagpal et al., 1998).
Both of these fingerprinting methods have been successfully applied in the differentiation of Bacillus spp., with some studies showing that RAPD-PCR offers a greater degree of resolution at strain-level compared to ITS-PCR (Logan et al., 2009; Martínez and Siñeriz, 2004; Daffonchio et al., 2000;
Daffonchio et al., 1998a; Daffonchio et al., 1998b).
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Identifying promising candidate BCAs at the taxonomic level is important when isolating and evaluating for biocontrol potential. This knowledge can offer insight into ecotypes amongst isolates, and can provide information as to the bacterial species extant in the chosen environment. Although 16S rRNA gene sequencing is widely regarded as the standard for bacterial characterisation, it is often insufficiently heterogenous to allow differentiation between closely-related species;
particularly in certain of the Bacillus groups of related taxa (Maughan and Van der Awera, 2011;
Daffonchio et al., 1998b). A range of alternate gene sequences have been used to differentiate AEFB species (Borriss et al., 2011; Rooney et al., 2009; Dickinson et al., 2004a; Reva et al., 2004; Roberts et al., 1994). Sequences of gyrase subunit A (gyrA) have shown sufficient sequence heterogeneity to allow closely-related members of the B. subtilis group to be distinguished and was therefore chosen for the current study (Chun and Bae, 2000).
Mass spectrometry has been widely used for the study of proteins and compounds produced by bacteria (Dare, 2006). Recently, MALDI-TOF-MS has been applied to the identification of bacteria (Lay, 2000). Whole bacterial cell preparations have been used to generate m/z peak lists to produce mass-fingerprints which have been used for identification purposes (Welker and Moore, 2011; Fox 2006). The mass spectrum is then compared to spectra within a database, using matching analysis software to provide an identity match (Welker and Moore, 2011). Conserved biomarker peaks in mass spectra can be specifically applied to identify bacteria at genus, species and strain levels (Carbonnelle et al., 2011; Lay, 2000; Wang et al., 1998). Cluster and inter-spectra analysis can also be applied to examine spectral diversity within an isolate set, or between isolates and reference strains (Fernández-No et al., 2013; Welker and Moore, 2011). Its ease-of-use and high-throughput has seen MALDI-TOF-MS applied for dereplication of bacterial isolates, assessments of genus- and species- level diversity within a set of isolates, and utilisation in taxonomic studies in microbiology (Ghyselinck et al., 2011; Welker and Moore, 2011).
Since Podosphaera fusca, is a biotrophic fungus, screening for BCA presents a challenge to researchers as the pathogen cannot be cultured under laboratory conditions without living host tissue. However, certain fungal species culturable on agar media have been applied as surrogates to screen for potential biocontrol candidates of cucurbit powdery mildew, and include: Botrytis cinerea, Fusarium oxysporum fsp. lycopersici, Rosellinia necatrix, Phytophthora cinnamomi and Penicillium digitatum (Romero et al., 2004). Consequently, surrogate pathogens have been applied in in vitro screening as a means of selecting for potential antagonists (Romero et al., 2004; Tewelde, 2004).
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A study was undertaken with the aim of isolating AEFB extant in the cucurbit phyllosphere and to screen them for antagonism of the foliar disease powdery mildew of cucurbits. Candidate AEFB isolates were assessed for antifungal activity in vitro using surrogate pathogens Rhizoctonia solani and Botrytis cinerea. Diversity amongst the selected isolates was then determined using RAPD- and ITS-PCR fingerprinting. Partial 16S rRNA and gyrA gene sequences were used to identify and differentiate selected isolates. The use of MALDI-TOF-MS to determine isolate diversity was also assessed.