1.5. Screening methods for AEFB as BCAs of cucurbit powdery mildew
1.5.2. Genetic fingerprinting to assess AEFB diversity
Reliability, time demands, and cost factors associated with phenotyping has resulted in bacterial differentiation based on metabolic characteristics being replaced by DNA-based means of bacterial strain differentiation (Li et al., 2009; Olsen and Woese, 1993). Currently, genotyping is widely used to distinguish between closely related bacterial species and strains because it is able to provide the high resolution required (Li et al., 2009). Methods of bacterial strain typing can be divided into three categories (Li et al., 2009):
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(i) DNA banding patterns separate strains by their DNA fragment sizes after amplification protocols or restriction enzyme digest (i.e. DNA fingerprinting);
(ii) DNA sequencing examines polymorphisms within a genomic DNA sequence; and (iii) DNA-DNA hybridisation uses DNA macro- and micro-array studies, whereby strains
are differentiated by hybridisation of probes to known sequences in the genome.
In prokaryotes there exist repetitive sequences spread throughout the genome, whose length and frequency are exploited as a means of fingerprinting (van Belkum, 1994). Alternately, tRNA sequences, protein structure genes, and gene expression regulators which occur frequently in the genome may be selected for (van Belkum, 1994). PCR-based genotyping studies rely on separation by gel electrophoresis to resolve the amplified DNA segments into their respective fingerprints (Olive and Bean, 1999). The resultant PCR amplicons will arise from either the presence of the specific target site or the distance between sites of primer annealing, depending on the type of PCR-primer protocol applied (van Belkum, 1994).
Factors considered in selecting a genetic fingerprinting technique include ease of interpretation, ease of application, level of technical difficulty, cost, and time required (Olive and Bean, 1999).
Within the scope of this study, methods applied for DNA fingerprinting of AEFB isolates were intergenic spacer region PCR (ITS-PCR) and randomly amplified polymorphic DNA PCR (RAPD-PCR), which will be further described below.
1.5.2.1. Fingerprinting of AEFB using RAPD-PCR
RAPD-PCR has been employed in microbiology for differentiation between bacterial isolates because it provides a high taxonomic resolution up to subspecies and strain levels. This technique has been successfully applied in the study of AEFB in both environmental and medical isolates (Kwon et al., 2009; Li et al., 2009; Rademaker et al., 2005; Nilsson et al., 1998; Tyler et al., 1997). RAPD-PCR fingerprinting employs randomly sequenced, DNA oligonucleotide primers of 9–10 base pairs in length, which will anneal to sites throughout the genome aided by non-stringent annealing temperatures (Li et al., 2009; Rademaker et al., 2005; van Belkum, 1994).
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Amplimers in RAPD protocols arise from two primers annealing at separate locations, resulting in a product the length between these two points (Li et al., 2009; Olive and Bean, 1999). The number and location of these random sites of compatible annealing throughout the genome are variable between bacterial strains, representing a means by which to discriminate between close relatives (Li et al., 2009; Ranjard et al., 2000; Olive and Bean, 1999). The migration patterns of the variable- length products are visualised on agarose or acrylamide gels (Li et al., 2009). In theory, the arbitrary primers will detect polymorphisms arising from deletions, mutations, or insertions to distinguish the strain or species by a distinct DNA fragment pattern (i.e. fingerprint) (Olive and Bean, 1999; Tyler et al., 1997).
Compared to other PCR-based strain differentiation methods, RAPD-PCR is fast, sensitive and relatively inexpensive (Li et al., 2009). Furthermore, the arbitrary primers are empirically designed and no previous knowledge of a target sequence is required (Rademaker et al., 2005). However, as RAPD is not targetted at any specific locus in the genome, it is more easily influenced by DNA and primer concentration, to the extent that changes in these variables may see amplification of new targets or the reduced amplification of others (Li et al., 2009; Olive and Bean, 1999; Tyler et al., 1997). Furthermore, RAPD-PCR works best with a pure DNA template (Rademaker et al., 2005).
The RAPD-PCR fingerprint patterns derived after electrophoresis may be complex and issues in interpretations may compromise the suitability of the method as a reliable and reproducible subtyping technique (Olive and Bean, 1999). Analysis of RAPD profiles has been greatly assisted by computer-aided analyses (Rademaker et al., 2005). Methods used in genotyping for strain differentiation require reproducibility to be fully trusted, and the main disadvantage to RAPD protocols is low reproducibility, making comparison among laboratories difficult (Li et al., 2009; Tyler et al., 1997). However, there are commercially available kits for RAPD-PCR which may assist in addressing some of this variability (Li et al., 2009). The worth of RAPD-PCR lies in its suitability for in- house comparisons of environmental bacterial strains, where resolution to strain level is highly valued for comparison and dereplication among a set of isolates (Logan et al., 2009; Rademaker et al., 2005).
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1.5.2.2. Fingerprinting of AEFB using ITS-PCR
The 16S ribosomal RNA (rRNA) subunit sequence is highly conserved in bacteria and therefore exhibits limited variability for the purposes of bacterial strain differentiation (Shaver et al., 2001).
This invariability necessitates the utilisation of non-coding sequences under less selection pressure than coding genes (Li et al., 2009). The intergenic spacers (ITS) (or intergenic spacer regions (ISR)) located between the 16S–23S rRNA subunit genes are sufficiently hypervariable in sequences and lengths to be suitable for bacterial strain typing (Daffonchio et al., 2003; Shaver et al., 2001;
Mileham, 1997).
ITS-PCR employs universal primers with 3’ ends facing outward from the 16S and 23S genes, resulting in a string of amplimers unique to a strain or species (Daffonchio et al., 1998a). ITS-PCR shows great promise in the discrimination of AEFB species (Dingman, 2012; Martínez and Siñeriz, 2004; Xu and Cote, 2003; Shaver et al., 2001). Bacillus and related genera have multiple ribosomal operons in their genomes, with members of the B. cereus group having as many as 12, and reference strain B. subtilis 168 possessing 10 operons (Daffonchio et al., 2003; Shaver et al., 2001). Within these operons the ITS lengths vary and, given variable mutations, the PCR amplification can generate species-specific banding patterns (i.e. fingerprints) after separation on low-resolution gel electrophoresis (Daffonchio et al., 2003; Shaver et al., 2001). However, this technique has been shown to fail to sufficiently differentiate between closely related species in a group, yet it is useful as a supplementary technique when different fingerprinting methods are being compared (Daffonchio et al., 1998a).