Sample collection and isolation
Potato tuber samples, displaying scab symptoms (as described by Gouws and McLeod (2012)), were collected from various potato production regions in South Africa. Isolation for Streptomyces from potatoes was done according to Loria and Davis (1989). The potatoes were washed and surface disinfested with 70% ethanol for 1 min. The potatoes were then rinsed thoroughly with sterile distilled water. The surface of the lesions was removed and discarded. The tissue under the scab lesion was cut into smaller pieces and crushed in 1 mL of sterile distilled water. Approximately 0.1 mL of the crushed tissue suspension was streaked onto Inorganic Salt Starch agar (ISSA - ISP medium 4 according to Shirling and Gottlieb (1966)). Plates were incubated at 28 °C for 5 days. Colonies resembling Streptomyces species were selected and purified by streaking on ISSA plates. Isolates were preserved as spore suspensions in 20% glycerol at -80 °C in the Streptomyces bacterial collection at the ARC-VIMP.
DNA extraction, PCR amplification and sequencing
Streptomyces isolates were grown on ISSA for 5 days at 30 °C, where after DNA was extracted for PCR and sequencing using the Zymo Research fungal/bacterial DNA isolation kit (Zymo Research Corporation, Irvine CA, USA). DNA was amplified by PCR using commonly used primers for bacterial characterisation (Bukhalid et al. 2002; Guo et al. 2008).
The PCR reactions consisted of 1 µL of template DNA, 1 µL of each primer, 12.5 µL of GC Tempase Mastermix II (Ampliqon A/S, Denmark) made up to 25 µL with PCR grade water. Conditions for the PCR included an initial denaturing at 95 °C for 15 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing of primers at 55 °C (for 16s rRNA), 63 °C (for rpoB and recA), 65 °C (for trpB), 60 °C (for atpD), and 58 °C (for gyrB) for 20 s, elongation at 72 °C for 30 s and a final extension step at 72 °C for 10 min. PCR products were visualized on 1% agarose gels, stained with ethidium bromide.
Forward and reverse strands of PCR products were sequenced by Inqaba Biotec (Pretoria, South Africa). The forward and reverse sequences were assembled and consensus sequences generated using CLC Genomics Workbench 10.0 (https://www.qiagenbioinformatics.com/). Sequences of type strains of Streptomyces species were downloaded from GenBank (https://blast.ncbi.nlm.nih.gov/) and aligned with newly generated sequences with the online version of MAFFTv.7 (http://mafft.cbrc.jp/alignment/server/index.html).
Phylogenetic analyses were conducted using PhyML 3.0 (Guindon and Gascuel 2003). Initial Maximum Likelihood trees of individual genes were constructed to check whether the different gene regions resulted in congruent tree topologies. After checking for congruence, the protein coding genes were combined into a single dataset. Selection of models of nucleotide substitution for the PhyML analyses, implementing the Akaike information criterion (AIC), was determined with jModeltest 2.1.7 (Guindon and Gascuel 2003; Darriba et al. 2012). Phylogenetic trees were mid-point rooted and bootstrap analysis was performed to determine branching point confidence intervals (1000 replicates). An initial analysis of 16S rDNA with all described Streptomyces species and all isolates obtained from fissure scab was conducted in order to determine which species are closely related to the fissure scab isolates. All subsequent analyses only included the selected subset of isolates.
Genome sequencing and annotation
Eight Streptomyces isolates were sequenced on Illumina HiSeq 2500 producing 2*125 bp paired-end reads. Quality control was done using FastQC v0.11.5 (Andrews 2010) and Trimmomatic v0.36 (Bolger et al. 2014). Genome assemblies were generated using SPAdes v3.12.0 (Bankevich et al. 2012) and the quality of the resulting assemblies evaluated by QUAST v4.6.3 (Gurevich et al. 2013). The assemblies were annotated with RAST (Overbeek et al. 2013). Average nucleotide identity (ANI) and average amino acid identity (AAI) matrices were calculated using the enveomics toolbox (Rodriguez-R and Konstantinidis 2016).
To elucidate possible mechanisms of pathogenicity, local BLAST searches for commonly found pathogenicity associated genes in Streptomyces were conducted on CLC Genomics Workbench. Annotated genomes were searched for virulence and pathogenicity associated genes.
Morphology and phenotypic analysis
Isolates from the three main clades identified by sequencing were selected for morphological investigation. Colony morphology was investigated on Yeast extract-Malt extract agar (YMA, ISP medium 2), Oatmeal agar (OA, ISP medium 3), ISSA, Gycerol-Asparagine agar (GA, ISP medium 5) and Tyrosine agar (TA, ISP medium 7). Isolates were streaked onto the different culture media, incubated at 30 °C in the dark and investigated for colony colour and pigment production after 7 and 14 days.
Spore chain morphology of isolates grown on OA for 7 days at 30 °C was investigated by scanning electron microscopy (SEM). Material was prepared by immersion in 2.5% glutaraldehyde in 0.075 M phosphate buffer (pH 7) for 1 h. The specimens were washed three times (10 min each) in 0.075 M phosphate buffer and fixed in 0.5% aqueous osmium tetroxide for 1–2 h. The material was washed three times in distilled water and dehydrated (10 min each) in 30%, 50%, 70%, 90%, and three times in 100%, ethanol. Hexamethyldisilizane (HMDS) was used to dry the material, where after it was mounted on aluminium stubs and coated with carbon. A Zeiss Crossbeam 540 FEG SEM was used to visualize the material.
Carbohydrate utilisation was determined using a 1% concentration of each carbon source (L-arabinose, D-fructose, D-glucose, I-inositol, D-mannitol, raffinose, rhamnose, sucrose and D-xylose) added to the carbon utilization medium (ISP medium 9 - Shirling and Gottlieb 1966) and rated for growth 14 days after incubation at 25 °C. The optimum temperatures for growth were assessed using ISP medium 1 at a range of 5–45 °C, with 5 °C intervals. Tolerance to sodium chloride was established using basal medium 5339 (10 g casein peptone L-1, 5 g yeast extract L-1, 15 g agar L-1) supplemented with 0–15% (w/v) sodium chloride with 2.5% intervals. The pH tolerance of isolates was tested on YMA plates at pH levels from 4 to 12. The pH levels of 4, 5.5, 7, 8.5 and 10.0 were adjusted with NaOH or HCl before autoclaving, and pH 11.5 was adjusted from pH 10 after autoclaving.
Screening of isolates by tuber slice assay
Initial screening of all Streptomyces isolates obtained from fissure scab symptoms were conducted to select virulent isolates for use in a greenhouse trial. Potato tubers cv. Mondial, were purchased from a supermarket in Pretoria. The tubers were washed thoroughly with water, disinfected in 1% sodium hypochlorite (NaClO) for 5 min, rinsed with sterile distilled water and air dried under a laminar flow hood. Inoculum was prepared for each isolate by making spore suspensions in sterile water in 20 mL McCartney bottles from mycelia and spores grown on ISSA plates for 5 days. Disinfected tubers were sliced into 7 mm thick slices with a sterilized knife and placed on top of moistened sterile filter paper in 90 mm plastic Petri dishes. A 10 µL aliquot of inoculum was pipetted in the centre of each tuber slice. Sterile water was included as a negative control. Petri dishes were placed in boxes lined with moist paper towels and incubated at 25 °C in the dark for 5 days. Treatments were replicated three times and arranged in a completely randomized design. The tubers were evaluated visually for the presence and size of the necrotic area on the tuber slice.
The pathogenicity of Streptomyces strains was investigated using two cultivars known to be susceptible to fissure scab, namely, Mondial and Innovator. Pots (25 cm diameter) were sterilized with 1% sodium hypochlorite and rinsed with sterile water. Compost was sterilized using a soil pasteurizer at 300 °C for 30 min and sterilized pots were filled halfway with the compost. One potato tuber was placed on top of the compost in each pot, after which the pots were filled with silica sand. The pots were maintained in a greenhouse at 25-28 °C and irrigated three times a week. Three isolates from each of the three main Streptomyces clades identified in the phylogenetic analysis were selected based on results from the tuber slice assay. The isolates were grown on ISSA for 7 days and scraped off to make a spore suspension to be used as inoculum.
At the tuber initiation stage, the pots were inoculated with the spore suspensions and placed in a randomized complete block design with 10 replicates. The control pots were inoculated with sterile distilled water. Potatoes were harvested three months after planting and evaluated for fissure scab symptoms using a custom rating scale:
Lesion length (LT):
1 = 1-25% of tuber length
2 = 25-50% of tuber length
3 = 50-75% of tuber length
4 = 75-100% of tuber length
Lesion depth (LD):
1 = lesion depth is superficial
2 = lesion depth is medium deep
3 = lesion depth is very deep
Scab Index = (LTxLD)/100
Streptomyces species were re-isolated from potatoes with fissure scab symptoms. The isolates were purified and identified in order to fulfil Koch’s postulates. Data were analysed with SAS software (SAS Institute, Inc., 1999) to determine significant differences between treatments.