AUDPC analysis of an agarized detached cotyledon assay evaluating nine AEFB isolates against pumpkin powdery mildew. AUDPC analysis of a biocontrol pot trial evaluating nine AEFB isolates against pumpkin powdery mildew.
Introduction
The screening of bacterial isolates for antagonism of powdery mildew of cucumbers is complicated by the biotrophic nature of the causal fungal pathogens, which require living host tissue to survive (Pérez-García et al., 2009). Screening approaches that can be used to select for AEFB as antagonists of cucumber powdery mildew.
The phyllosphere habitat and foliar biocontrol
Plant cell leakage and secretory organs such as trichomes and hydathodes provide most of the nutrients available to the microflora, although exogenous sources such as pollen or honeydew are seasonally available (Blakeman and Fokkema, 1982). Because of the more transient and exposed nature of the phylloplane, biocontrol of plant diseases has generally been more successful in the rhizosphere than in the phyllosphere (Vorholt, 2012; Andrews, 1992).
Bacillus and related genera in plant health and disease control
Bacillus spp. lipopeptide compounds and their roles in plant disease control
Lipopeptide compounds consist of a cyclic or short linear oligopeptide linked to a lipid tail (Raaijmakers et al., 2010). It has been demonstrated that lipopeptide compounds have different roles in relation to plants (Pérez-García et al., 2011).
Powdery mildew on cucurbits
Cucurbit powdery mildew life cycle and epidemiology
Life cycle of Podosphaera fusca, the primary causative agent of pumpkin powdery mildew (Pérez-García et al., 2009). The chasmothecium is the result of sexual reproduction in both fungi and is generally perceived as a source of overwintering and summer inoculum (Pérez-García et al., 2009).
Control measures for powdery mildew of cucurbits
- Bacillus subtilis and powdery mildew biocontrol
The requirement of lipopeptides for cucurbit powdery mildew antagonism was further supported by the lack of disease antagonism after site-directed mutagenesis of a single site in the bacterial genome that controls production of key lipopeptides (Romero et al., 2007a). The researchers concluded that effective bacterial colonization is the key to successful biocontrol of pumpkin powdery mildew, and that active vegetative cells provided effective and persistent antifungal activity (Romero et al., 2007c).
Screening methods for AEFB as BCAs of cucurbit powdery mildew
- Isolation and characterisation of AEFB
- Genetic fingerprinting to assess AEFB diversity
- Fingerprinting of AEFB using RAPD-PCR
- Fingerprinting of AEFB using ITS-PCR
- DNA sequence polymorphisms for AEFB differentiation
- Antifungal activity screening and assessment of powdery mildew antagonism
- Lipopeptide compounds analysis and production potential in AEFB
- Extraction and analysis of lipopeptide compounds
- Gene marker PCR to determine lipopeptide production potential
- MALDI-TOF-MS for bacterial identification
Migration patterns of products of variable length are visualized on agarose or acrylamide gels ( Li et al., 2009 ). The analysis of RAPD profiles has been greatly aided by computer-assisted analysis (Rademaker et al., 2005).
Conclusion
Replicate mass spectra are required for accurate interpretation and pattern recognition, therefore spectral reproducibility is important for microbial characterization (Dare, 2006; Wunschel et al., 2005; Keys et al., 2004; Lay, 2001; Wang et al., 1998). Furthermore, the strain-level resolution achievable with MALDI-TOF-MS is higher than the more commonly used DNA electrophoresis or other protein profiling methods ( Ghyselinck et al., 2011 ; Welker and Moore, 2011 ).
Introduction
DNA fingerprinting is often used as a means to determine strain diversity among a set of bacterial isolates (Ghyselinck et al., 2011; van Belkum, 1994). Consequently, surrogate pathogens have been applied in in vitro screening as a way to select for potential antagonists (Romero et al., 2004; Tewelde, 2004).
Materials and Methods
- Isolation of AEFB from cucurbit leaf material
- Assessment of antifungal activity of AEFB using dual-culture bioassays
- DNA sequencing of 16S rRNA and gyrase subunit A gene fragments
- AEFB classification using MALDI-TOF-MS
The outgroup sequence for the phylogenetic tree of the 16S rRNA gene sequence was Staphylococcus pasteuri ATCC51129 (NR024669.1). Phylogenetic trees for 16S rRNA and gyrA gene fragment sequences were generated using MEGA6 software (version 6.0) (Tamura et al., 2013) and aligned using MUSCLE in MEGA6 (Edgar, 2004).
Results
In vitro screening for antifungal ability using dual-culture bioassays
Double culture antifungal bioassays illustrating different levels of AEFB antagonism against Botrytis cinerea and Rhizoctonia solani on PDA after 4 days of incubation at 30°C. Effect of dual-culture antifungal bioassay duration on fungal antagonism of Botrytis cinerea by isolate bng119, illustrating the initiation of fungistatic interactions over a 14-d period. Results for double-culture antifungal bioassays of selected AEFB isolates against Botrytis cinerea and Rhizoctonia solani.
Observations of AEFB isolate colony morphology, cell morphology, and sporangial
When Gram stained, it was confirmed that all AEFB isolates were Gram-positive rods capable of producing endospores (Table 2.4). The location of the forming endospores within the sporangium was predominantly subterminal, but centralized endospore locations were observed for some isolates. Most isolates showed no swelling of the parent cell by the forming spores, although four isolates showed noticeable sporangial swelling, while 19 isolates showed mild swelling.
Fingerprinting of AEFB isolates using RAPD-PCR and ITS-PCR
Fifty-five phyllosphere-isolated AEFB isolates from the initial dual-culture bioassays were subjected for DNA fingerprinting using ITS-PCR and RAPD-PCR. These isolates were selected primarily on the basis of their antifungal activity against one or both of the test fungi screened in the dual culture bioassays, and were representative of each location and cucumber species sampled.
RAPD-PCR differentiation of phylloplane-isolated AEFB isolates
Fingerprint profiles of AEFB isolates from RAPD-PCR with OPG-11 primer as seen by electrophoresis using 1.5% agarose gel. The RAPD-PCR fingerprints of each of the reference strains used in this study are shown in Figure 2.3. RAPD-PCR fingerprint profiles of selected AEFB reference strains generated with primer OPG-11 as seen by electrophoresis using a 1.5% agarose gel.
ITS-PCR differentiation of phylloplane-isolated AEFB isolates
Fingerprint profiles of AEFB isolates from ITS-PCR as seen after electrophoresis with 1.5% agarose gel. ITS-PCR fingerprint profiles of selected AEFB reference strains, as seen after electrophoresis with 1.5% agarose gel. From RAPD and ITS-PCR fingerprint results (Table 2.7), it was observed that fingerprint profiles were neither host plant nor region specific.
Sequencing of 16S rRNA gene fragment
Representative isolates were chosen for sequencing 16S rRNA and gyrA gene fragments based on fingerprint grouping, cucurbit host species, and geographic sampling location. 16S rRNA gene sequence data from the AEFB isolates and reference strains from GenBank were used to infer phylogenetic relationships (Figure 2.8). Comparative matches of AEFB isolates following a BLAST search on GenBank of the 16S rRNA subunit gene fragment after sequencing (accessed: March 20, 2015).
Sequencing of gyrA gene fragment
Comparative matches of AEFB isolates after a BLAST search on GenBank of the gyrA gene fragment after sequencing (date accessed: 20 March 2015). The gyrA gene sequence data from the AEFB isolates and reference strains obtained from GenBank were used to infer phylogenetic relationships (Figure 2.10). A greater degree of sequence heterogeneity was evident in the gyrA phylogenetic tree than for the 16S rRNA sequence phylogenetic tree (Figure 2.8).
Applying MALDI-TOF-MS to differentiate AEFB isolates
ˠ Clusters of RAPD-PCR fingerprint profiles described in Figure 2.2 * Sequence matches from 16S rRNA and gyrA gene fragment sequencing. From these MSPs, a dendogram was generated to reflect the relationship between the isolates based on variances in their mass spectra (Figure 2.10). Another dendogram was generated incorporating AEFB reference strains from the Biotyper database, as shown in Figure 2.12.
Discussion
Sequence analysis of the 16S rRNA gene is considered a benchmark for bacterial isolate identification and diversity assessment (Woo et al., 2008; Goto et al., 2000). MALDI-TOF-MS was evaluated in this study as a means of rapidly identifying isolates, as its resolution is considered to be similar to that achievable by 16S rRNA gene sequence analysis and RAPD-PCR fingerprinting (Welker and Moore, 2011; Dickinson et al. et al., 2004b). Use of MALDI-TOF-MS has been reported to provide a simpler and faster method for characterizing isolates than sequencing and is able to achieve levels of sensitivity comparable to 16S rRNA sequence analysis (Ghyselinck et al., 2011).
Introduction
Many AEFB lipopeptide variants are believed to play a range of roles within the plant-related environment (Ongena et al., 2010; Ongena et al., 2009; Ongena and Jacques, 2008). Iturines are believed to play an important role in the spread of bacteria; antifungal activity and stimulation of plant host resistance (Ongena et al., 2009; Ongena and Jacques, 2008). The fengycins are believed to play a role in stimulating plant host resistance and exhibit antifungal properties (Ongena and Jacques, 2008; Ongena et al., 2007; Ongena et al., 2005b).
Materials and Methods
- PCR detection of gene markers associated with lipopeptide production
- Lipopeptide compound extraction from Bacillus cultures
- Determination of antifungal activity of methanol extracts using a disc-diffusion bioassay
- MALDI-TOF-MS analysis of lipopeptide-methanol extracts
For the purposes of lipopeptide compound extraction, selected Bacillus isolates were grown in a defined antibiotic production medium (McKeen et al., 1986). To determine the antifungal activity of the methanol extracts, disc diffusion bioassays were performed using Rhizoctonia solani obtained from the Discipline of Plant Pathology (University of KwaZulu-Natal) culture collection. TLC analysis of the methanol extracts was performed on an oven-dried TLC Silica Gel 60 F254 aluminum plate (20 cm x 20 cm) (Merck, Germany).
Results
PCR detection of gene markers associated with lipopeptide biosynthesis
Results for screening isolates for lipopeptide gene markers are shown in Table 3.4, with examples of positive bands shown in Figure 3.2. Isolate bng241 did not give any positive results for the examined gene markers, which was not unexpected due to the lack of antifungal activity of this isolate. Lipopeptide gene markers associated with fengycin (fenD), bacillomycin (bacC), surfactin (sur3), and iturin (ituD) biosynthesis among Bacillus isolates compared to RAPD fingerprint clustering.
Extraction and characterisation of lipopeptide compounds from Bacillus isolates
- Disc-diffusion bioassay
- Analysis of methanolic extracts using TLC
- Detection of lipopeptide compounds using MALDI-TOF-MS
Methanolic extracts of the selected isolates were also analyzed by MALDI-TOF-MS to screen for lipopeptide biomarkers. Detection of lipopeptide biomarkers in methanolic extracts of Bacillus isolates using MALDI-TOF-MS analysis. Comparative data of lipopeptide PCR gene markers and MALDI-TOF-MS detection of lipopeptide compounds in methanolic extracts of Bacillus isolates.
Discussion
Disparity between the iturin and bacillmycin gene marker PCR data was further highlighted by MALDI-TOF-MS analysis data (Table 3.8). All isolates positive for the bacillomycin gene marker were confirmed as bacillomycin producers when extracts were analyzed using MALDI-TOF-MS extract analysis. However, differences in the synthetic ability of these two species were revealed by MALDI-TOF-MS analysis of.
Introduction
Two methods, namely an agarized detached cotyledon assay and a biocontrol pot experiment, were used to evaluate disease antagonism.
Materials and Methods
- Bacterial isolates
- Cultivation of powdery mildew disease and conidia harvesting
- Antagonism screening using an agarised detached cotyledon bioassay
- Biocontrol pot trial screening of Bacillus isolates against powdery mildew
- Impacts of inoculum preparation on cucurbit powdery mildew antagonism
Examples of leaf area infected (l.a.i.) metrics used for grading powdery mildew of pumpkin disease in the biocontrol pot trial. Impacts of inoculum preparation on cucumber powdery mildew antagonism The impact of bacterial inoculum preparation of P. powdery mildew susceptible patty pan host plants (Yellow Scallop hybrid STAR 8081) (Starke-Ayres (Pty) Ltd., South Africa) was in grown pots (18 cm diameter) filled with composted pine bark medium (~2.3 L volume).
Results
Agarised detached cotyledon assay
The non-disease control was housed in a separate growth chamber under the same conditions as the experimental treatments. Disease reduction percentages were expressed as a function of the treatment AUDPC value and the diseased control AUDPC value. The diseased controls for the detached cotyledon test showed initial symptoms of powdery mildew infection within 5 days of fungal inoculation, after which symptoms progressed rapidly.
Biocontrol pot trial of powdery mildew antagonism
Development of powdery mildew disease on squash (Parthenon hybrid F1, Starke-Ayres, South Africa) inoculated with B. No bacterial treatment provided a statistically significant reduction in disease compared to the diseased control.
Impacts of bacterial preparation and culture age of B. amyloliquefaciens strain cce175
Only treatments 3 and 4 offered any disease reduction, although this disease reduction was significantly lower than that achieved with fungicide control at 8.14% and 1.34%, respectively. The number of conidia/cm2 of leaf material varied between treatments and was not correlated with the calculated AUDPC values (Table 4.4). Interestingly, treatment 3 (48-hour cell-free supernatant) provided the highest fungal spore count at 3.91 x 105 spores/cm2 of leaf material.
Discussion
Efficacy of Bacillus subtilis and Ampelomyces quisqualis biocontrol agents used in combination with fungicides against squash powdery mildew. The effect of lipopeptides of antagonistic strains of Bacillus subtilis on the morphology and ultrastructure of the cucurbit pathogen Podosphaera fusca. Phylogeny and molecular taxonomy of the Bacillus subtilis species complex and description of Bacillus subtilis subsp.
