Microbiology

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Investigation into the diversity of antifungal aerobic endospore-forming bacteria associated with bulk and crop rhizosphere soil.
(2011) Musoke, Jolly.; Hunter, Charles Haig.
Members of the genus Bacillus are mainly Gram positive, aerobic rod shaped, endospore-forming bacteria that are increasingly being recognised for their ability to promote plant growth and antagonise fungal pathogens. From a biological control perspective, Bacillus spp. strains that produce antifungal compounds are of particular interest. In this study, aerobic endospore-formers were isolated from an undisturbed indigenous grassland soil and screened for antifungal activity and other plant growth promoting traits. Endospore-formers were also isolated from rhizosphere soil associated with the roots of maize, wheat and kale grown in pots containing soil from the same grassland site. Microbial diversity amongst isolates showing antifungal activity was investigated using different molecular fingerprinting methods, namely, intergenic transcribed spacer–PCR (ITS-PCR), random amplified polymorphic DNA-PCR (RAPD-PCR) and 16S rRNA gene amplification and sequencing. Characterization of the active antimicrobial compound(s) associated with selected isolates was also attempted. Prior to isolating from bulk and rhizosphere soils, samples were pre-heated to eliminate heat sensitive vegetative cells. Mean endospore counts were; wheat rhizosphere, Log 6.03 c.f.u g-1 soil; maize rhizosphere, Log 5.88 c.f.u g-1 soil; kale rhizosphere Log 5.90 c.f.u g-1 soil; and bulk soil Log 5.67 c.f.u g-1soil. A total of three hundred and eighty-four isolates were screened for antagonism towards Rhizoctonia solani using dual-culture plate bioassays. Thirty four of the isolates (~9%) mostly isolated from the bulk soil inhibited R. solani at varying degrees. Differences in antimicrobial interactions were apparent in in vitro bioassay; supposedly due to different concentrations and/or types of antimicrobial compounds. Biochemical tests for amylase, cellulase, chitinase, and proteinase activity, siderophore production and inorganic phosphate solubilisation were conducted. None of the isolates possessed all of these attributes and only a few showed multiple traits. Ninety-one percent of the isolates exhibited proteinase activity, 76% were able to hydrolyze starch whereas only four displayed cellulase activity. Only four isolates from the bulk-soil were capable of solubilising inorganic phosphate. ITS-PCR and 16S rRNA gene sequence analysis showed high levels of genetic homology amongst isolates and the majority were closely associated with representatives of the B. cereus group. Isolate C76 was the exception, being closely matched with B. subtilis. ITS-PCR banding profile was useful for distinguishing between species but did not distinguish within species. RAPD-PCR distinguished finer levels of genetic diversity between and within sample sets, with primer OPG-11 showing the greatest levels of heterogeneity. DNA extraction methods and the influence of template DNA dilution were investigated to determine their influence on RAPD-PCR analysis reproducibility. Prominent bands were comparable for crude template- and kit-extracted DNA but slight changes in band intensity and in some instances, additional faint bands were observed. At the highest DNA concentrations tested (7 μg/ml), further bands with molecular weights above 2.5 kbp were apparent. Strict standardization of PCR conditions greatly reduced variability of the RAPD-PCR analysis. Isolates from the different sample sets were screened for the presence of genetic markers associated with the biosynthesis of zwittermicin A, an aminopolyol antibiotic produced by some members of the B. cereus group. In an initial screen only one isolate, W96, yielded PCR amplicons consistent with those previously reported in the literature for the zwittermicin A genes. Later a further sixteen isolates grouped with W96 on the basis of the RAPD-PCR fingerprinting profiles, were screened for the presence of these genes. Of these, only six showed PCR amplification products similar to W96. Sequence homology testing against the GenBank database confirmed the presence of the zwittermicin A genes in these isolates. Isolate W96 was selected for further extraction and characterization of its antifungal compound(s). However, after culturing in various broth media cell free supernatants of W96 failed to show antifungal activity in vitro even when the supernatants were concentrated 20-fold. These findings provide a general overview of the diversity of aerobic endospore-forming bacteria present in an undisturbed indigenous grassland soil that exhibited antifungal activity in vitro and the limited influence tested crop rhizospheres have on this diversity. Combined use of ITS-PCR, 16S rRNA sequencing and RAPD-PCR techniques served as a rapid and effective means of grouping isolates for further investigations of their potential use as biocontrol agents and plant growth promoting rhizobacteria.
Screening for aerobic endospore-forming bacteria as biocontrol agents for powdery mildew disease of cucurbits.
(2015) Tredgold, Heather Rayne.; Hunter, Charles Haig.
Powdery mildew of cucurbits costs the South African cucurbit-growing industry millions of Rands per year in reduced yields and compromised fruit quality. Amongst the many bacterial and fungal antagonists of cucurbit powdery mildew, certain aerobic endospore-forming bacteria (AEFB) species show promise as biocontrol agents of this disease. When embarking upon biocontrol agent selection, multifaceted screening strategies are crucial. A study was undertaken with the aim of isolating AEFB from the cucurbit phylloplane for evaluation as potential antagonists of cucurbit powdery mildew using various screening approaches. Three hundred and nine AEFB isolates were isolated from cucurbit leaf material sourced from eight locations in the greater Msunduzi, KZN region. Dual-culture antifungal bioassays were performed using surrogate phytopathogenic fungi Botrytis cinerea and Rhizoctonia solani in place of the obligately biotrophic Podosphaera spp.. Two PCR-based genotyping methods were used to differentiate and group 55 antifungal AEFB isolates: internal-transcribed spacer region (ITS) PCR and randomly amplified polymorphic DNA (RAPD) PCR. The RAPD-PCR distinguished greater levels of genetic polymorphisms amongst isolates than did the ITS-PCR, revealing 14 different profiles as opposed to the three obtained from ITS-PCR; with 42% of isolates associated with a single RAPD-PCR banding profile. Phylogenetic relationships between representatives of each of the RAPD-PCR fingerprint groupings were determined by sequence analysis of 16S rRNA and gyrase subunit A (gyrA) gene fragments. In each instance, several distinct clusters were discernable, though gyrA sequences displayed higher levels of strain-level sequence heterogeneity. Comparisons of both gene sequence types with reference strains from the GenBank database revealed similarities to several known plant-associated strains of AEFB, including B. amyloliquefaciens subsp. plantarum and B. subtilis. Matrix-assisted laser deionisation-desorption time-of-flight mass spectrometry (MALDI-TOF-MS) based identification of selected AEFB was evaluated by comparing spectral data from AEFB isolates with reference strains in a Bruker BDAL Biotyper database. Only three out of the 14 isolates evaluated were identified to species level with acceptable confidence levels. This poor taxonomic resolution was ascribed to a paucity of applicable reference strains in the BDAL library. Nevertheless, mass spectra profiles of each isolate allowed for the clustering of related isolates to be achieved when dendograms were created. Antifungal compounds were extracted from 14 isolates using an acid-precipitation and methanol extraction protocol. Detection and identification of lipopeptide compounds in these extracts was assessed using thin-layer chromatography (TLC) and MALDI-TOF-MS. PCR-based screening for lipopeptide production potential using selected lipopeptide gene markers (viz. surfactin, iturin, bacillomycin, and fengycin) was also evaluated for the selected 14 isolates. These isolates were found to produce multiple lipopeptide compounds; including homologues of surfactin, iturin, and fengycin. However, disparities that emerged between PCR, TLC, and MALDI-TOF-MS data suggest that some PCR primers, the ituD marker in particular, showed limited specificity amongst the AEFB strains screened. Based on the overall findings, nine isolates proceeded to in vivo screening against Podosphaera spp. using an agarised detached cotyledon assay and a biocontrol pot trial. Isolates achieving the most effective antagonism of Podosphaera spp. differed in each respective assay. Isolate cce175 provided the highest antagonism in the biocontrol pot trial, and isolate sqo279 provided the best results in the detached cotyledon assay. The impacts of inoculum preparation were assessed using isolate cce175 in a biocontrol pot trial. Treatments varied in cell growth phase and assessed cell-free supernatant, whole broth, and cell-only fractions on biocontrol efficacy compared to a Tebuconazole (430 g/l) fungicide control. None of the treatments were found to impact disease at a statistically significant level. The merits and limitations of the various screening approaches used, and issues surrounding the isolation and assessment of biocontrol efficacy in plant-associated AEFB, are discussed.
Screening of aerobic endospore-forming bacterial isolates as candidate biocontrol agents against rhizoctonia solani.
(2016) Hunter, Charles Haig.; Schmidt, Stefan.; Laing, Mark Delmege.; Wallis, Frederick Michael.
Bacterial-based biocontrol of soil-borne phytopathogens has gained prominence as a promising technology for developing sustainable agricultural pest control practices. Aerobic endospore-forming bacteria are seen as potential candidates for biocontrol applications due to various ecological and physiological traits which have been shown to influence plant health and disease control. Their ability to produce endospores also provides a major commercial advantage over non spore-forming bacteria. Appropriate screening methods are central to the discovery of successful biocontrol agents and should ideally be both ecologically relevant and able to evaluate a large number of isolates. A study was therefore undertaken with the aim of establishing screening methods that facilitate the selection of aerobic endospore-forming bacteria as candidate biocontrol agents against Rhizoctonia solani, an economically important fungal pathogen exhibiting a wide host range. Aerobic endospore-forming bacteria were isolated from rhizosphere material of five crop types grown in composted pine bark medium and screened for R. solani antagonism using traditional in vitro dual-culture bioassays. Isolates exhibiting antifungal activity were then evaluated in vivo for biocontrol activity against R. solani in cucumber seedling trials. Selected isolates were evaluated further using several screening approaches including: genomic fingerprinting; characterization of, and PCR-based screening for genes involved in the biosynthesis of bioactive lipopeptide compounds; and, the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a means of rapidly screening bacterial isolates. Approximately 6% of the bacterial isolates (n=400) showed antagonism towards R. solani in vitro. Dual-culture bioassays against R. solani, Fusarium oxysporum, Botrytis cinerea and Pythium arrhenomanes revealed that the antagonistic activity amongst isolates varied considerably and was influenced by the duration of the assay. From these assays it was possible to rank isolates based on the extent and stability of the inhibitory response in vitro as well as by the spectrum of antifungal activity observed. Twenty-four isolates were selected for in vivo screening for biocontrol activity against R. solani, using susceptible cucumber plants grown under greenhouse conditions. In preliminary experiments the pathogen loading rates were shown to have a marked influence on disease severity. In experiments where R. solani was seeded in the form of colonized agar plugs, significant differences between treatments and controls were recorded and several potential biocontrol candidates were identified. A general observation was that isolates that achieved high rankings in vitro performed better in the in vivo trial than those with lesser rankings; although some exceptions were noted. These findings support the notion that fungal antagonism is an important determinant of biocontrol potential that can be used in preliminary biocontrol screening programmes. Internal-transcribed spacer region (ITS) PCR and randomly amplified polymorphic DNA (RAPD) PCR were evaluated as methods to differentiate isolates exhibiting antifungal activity in vitro. ITS-PCR distinguished three major groupings, but proved to be limited in its ability to detect inter- and intra-specific variation amongst closely related organisms. Based on 16S rRNA gene sequence analysis, two of the groups were identified as members of the “Bacillus subtilis” and “Bacillus cereus” clusters; while, the third group consisted of a single isolate identified as a strain of Brevibacillus laterosporus. RAPD-PCR revealed further levels of genetic diversity within each ITS grouping. The “Bacillus subtilis” cluster was distinguished further into four distinct groups, which based on gyrA gene fragment sequence analysis, were identified as strains of B. amyloliquefaciens subsp. plantarum and B. subtilis respectively. Sequence matches were consistent with the RAPD-PCR groupings, indicating that this method was appropriate for differentiating related isolates at the strain and possibly the sub-species level. Clonal similarities were evident for a number of strains isolated from different plant species suggesting that these may reflect populations of rhizosphere competent strains and/or plant adapted ecotypes. Strains of B. amyloliquefaciens subsp. plantarum and B. subtilis were amongst the best performers in the in vivo biocontrol seedling trial and generally performed better than the “Bacillus cereus” group of isolates. RAPD-PCR of the “Bacillus cereus” isolates showed that they exhibited greater levels of genetic heterogeneity and that the groupings detected were not consistent when different primer sets were evaluated. Genomic fingerprinting was found to provide an insight into the prevalence, distribution and possible rhizosphere competency of related strains. Liquid chromatography was used in conjunction with electrospray-ionization time-of-flight (ESI-TOF) mass spectrometry (MS) to characterize bioactive lipopeptides purified from culture supernatants of selected strains that ranked highly in the in vitro/in vivo assays. Phylogenetically related strains produced very similar lipopeptide profiles. Bacillus subtilis strains were found to produce isoforms of surfactin and fengycin. In addition to these lipopeptides, B. amyloliquefaciens subsp. plantarum strains were also found to produce isoforms of bacillomycin D or iturin A. Bacillomycin/iturin and fengycin fractions exhibited antifungal activity in vitro, whereas surfactin fractions did not. Isolates that ranked the highest in the R. solani dual-culture bioassays all produced either isoforms of bacillomycin D or iturin A. Bacillomycin D producing isolates were amongst the best performers in the in vivo biocontrol trials. Gene markers targeting the biosynthetic apparatus of the detected lipopeptide classes were then assessed for screening purposes using PCR. BACC1F/1R primers targeting the bacillomycin D synthetase C (bmyC) gene correlated well with the ESI-TOF MS findings, whereas ITUD1F/1R primers targeting the malonyl-CoA-transacylase (ituD) gene linked to iturin A biosynthesis were unable to distinguish between isolates that produced iturin or bacillomycin in culture. Disparities between some of the PCR and ESI-TOF MS results suggested that primers targeting srfA (surfactin) and fenD (fengycin) biosynthetic genes showed limited specificity amongst the strains screened. Phylogenetic comparisons of srfD and fenD gene sequences from selected strains of B. amyloliquefaciens subsp. plantarum and B. subtilis revealed that these genes clustered according to species with marked heterogeneity between clusters being evident. Using fenD gene sequence data from B. amyloliquefaciens subsp. plantarum FZB42, primers (FENG1F/1R) targeting fengycin synthetase genes of strains of B. amyloliquefaciens subsp. plantarum isolated in this study were successfully established. MALDI-TOF MS was assessed as a means of identifying isolates antagonistic to R. solani in vitro and determining their associated lipopeptide profiles. Mass spectra were obtained in the m/z range 2000 to 20000 for identification and grouping purposes and in the m/z 750 to 2500 range in order to profile lipopeptide production. The available Bruker BDal spectral library allowed for the identification of isolates to the genus level but proved to be limited for identifying environmental isolates to the species level. Extension of the library using “inhouse” mass spectra generated from isolates identified in this study significantly improved the level of isolate identification in subsequent identification runs. Cluster analysis of mass spectra allowed for the relationships between isolates to be established and provided a means of grouping closely related isolates. Strains of B. subtilis and B. amyloliquefaciens were clearly distinguished from one another and the potential for differentiating strains at the subspecies level was also shown. MALDI-TOF MS also provided a convenient means of detecting bioactive lipopeptides directly from whole cell preparations, cell extracts and crude culture filtrates. Lipopeptide profiles varied depending on taxonomic groupings. Results for isolates within the “Bacillus subtilis” group supported the earlier ESI-TOF MS findings and were found to be more reliable than PCR screening for lipopeptide synthesis genes. “Bacillus cereus” group isolates produced distinct spectral profiles with peaks that were consistent with biomarkers previously described in the literature as isoforms of the kurstakin class of lipoheptapeptides. Brevibacillus laterosporus CC-R4 yielded a unique spectral profile in the m/z 750-2000 range with mass fragments which were similar to antimicrobial compounds recently reported in the literature. Overall, MALDI-TOF MS was found to fulfil the requirement for a practical yet robust technique suitable for processing large numbers of aerobic endospore-forming bacteria for biocontrol screening. This study has shown that genomic fingerprinting and MALDI-TOF MS characterization of bacterial isolates are worthwhile additions to preliminary in vitro screening practices. They provide a level of isolate differentiation and characterization that is beneficial for selecting candidate biocontrol agents, which is not possible with traditional screening practices. Effectively, they allow traditional biocontrol screening to move away from empirically based approaches to ones which are “knowledge” based, allowing for representative groups of bacteria with specific traits to be selected for further evaluation.

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