Streptomyces Species Associated with Fissure Scab of Potato in South Africa Including Description of S. Resiliuntiscabiei Sp. Nov.

Streptomyces species are the causal agents of several scab diseases on potato tubers. A new type of scab symptom, caused by Streptomyces species, was observed in South Africa from 2010 onwards. The disease was initially thought to be caused by a single Streptomyces species, however, subsequent isolations from similar symptoms on other potato tubers revealed diversity of the Streptomyces isolates. The objective of this study was to characterise these isolates in order to determine which species are involved in the disease. This was done by sequencing and phylogenetic analyses of the 16S rDNA as well as ve housekeeping genes, investigation of growth on different culture media, standard phenotypic tests and scanning electron microscopy of culture morphology. The presence of the pathogenicity island (PAI) present in plant pathogenic Streptomyces species was also investigated. The genomes of eight isolates selected from the three main clades identied, were sequenced and annotated to further clarify species boundaries. Two isolates of each of the three main clades were also inoculated onto susceptible potato cultivars in order to establish the pathogenicity of the species. The results of the phylogenetic and genome analyses revealed that there are three main species involved, namely, S. werraensis, S. pseudogriseolus and a novel Streptomyces species that is described here as Streptomyces resiliuntiscabiei sp. nov. The glasshouse trial results showed that all three of the Streptomyces species are capable of producing ssure scab symptoms. None of the Streptomyces isolates from ssure scab contained the full PAI and the mechanism of disease initiation still needs to be determined.


Introduction
Streptomyces species are causal agents of some of the most wide-spread and economically important diseases in potato production worldwide (Braun et al. 2017;Wanner 2006Wanner , 2009). Common scab is caused by species in the Streptomyces scabiei complex. Other related Streptomyces species are the causal agents of netted scab in Europe (Scholte and Labruyére 1985) and russet scab in North America and Japan (Faucher et al. 1993; Oniki et al. 1986). All of these diseases are caused by complexes of Streptomyces species and have large variations in symptom expression (Bouchek-Mechiche et al. 2000;Wanner 2006Wanner , 2009. Plant pathogenicity in the Streptomyces genus has been proven to be based on a speci c toxin, thaxtomin, which is produced by pathogenic strains causing common scab. Genes encoding enzymes for the synthesis of thaxtomin (txtA, txtB, txtC, and nos) are located in a pathogenicity island (PAI) (Loria et al. 2006). Other genes encoding virulence factors also found on the PAI in pathogenic Streptomyces species include tomatinase (tomA) and the necrosis-inducing protein (nec1) (Wanner 2006). Thaxtomin inhibits cellulose biosynthesis and is able to induce plant cell hypertrophy in growing plant tissues (Fry and Loria 2002;Scheible et al. 2003), thereby causing the scabs on potato tubers (Fiers et al. 2012). Tomatinase is responsible for plant defence suppression (Kers et al. 2005) by detoxi cation of tomatin, an anti-microbial saponin (Roldán- Arjona et al. 1999). The nec1 gene encodes a small necrogenic protein with an unknown plant cell target (Bukhalid et al. 1998) that may be involved in plant defence suppression (Joshi et al. 2007).
The availability of whole genome sequences for thousands of organisms have enabled researchers to study elds like the evolution of organisms (Chandra and Chater 2014 ) Bukhalid et al. (2002) also presented evidence of HGT between Streptomyces species causing the emergence of multiple plant-pathogenic species. This mechanism can therefore lead to non-pathogens acquiring pathogenicity genes and causing diseases that previously did not exist.
The genus Streptomyces has undergone extensive revisions and currently contains around 800 species (http://www.bacterio.net/streptomyces.html), of which most are saprophytic and present in large numbers in the soil (Janssen 2006). There have been numerous name changes and taxonomic revisions of the species in this genus since the rst description of the common scab pathogen as Oöspora scabies by Thaxter in 1892 (Güssow 1914). One of the most comprehensive reviews was initiated by the International Streptomyces Project (ISP) committee, which was established in 1963. This provided a standard set of tests to use in describing Streptomyces species (Shirling and Gottlieb 1966 Parks et al. 2018), these methods have been superseded in the description of bacterial species. However, many Streptomyces descriptions still include results of biochemical tests, as these may be an indication of useful secondary metabolites produced by the Streptomyces species.
In South Africa, Gouws and McLeod (2012) described a new disease of potatoes, associated with Streptomyces species. The symptoms on potato tubers consisted of deep longitudinal cracks with scab-like lesions (Fig. 1a), and was termed " ssure scab". These blemishes differ from common scab (Fig. 1b) and growth cracks (Fig. 1c) in the star shaped ssures that is commonly associated with ssure scab.
The causal agent was identi ed as a Streptomyces species related to S. vinaceus, S. malachiticus, S. werraensis, S. cyaneus and S. pseudogriseolus. The four isolates that were sequenced during the initial description of the disease were identical (Gouws and McLeod 2012), leading researchers to believe that a single Streptomyces species was responsible for the disease. However, subsequent isolations from ssure scab symptoms over the last eight years have revealed considerable morphological variation in the Streptomyces isolates obtained from these symptoms. Further investigations into the causal agents of ssure scab was therefore initiated and the results of these studies are presented here.

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 puri ed 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 ampli cation 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 ampli ed 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 nal 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
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 con dence intervals (1000 replicates). An initial analysis of 16S rDNA with all described Streptomyces species and all isolates obtained from ssure scab was conducted in order to determine which species are closely related to the ssure scab isolates. All subsequent analyses only included the selected subset of isolates.
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 identi ed 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 xed 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, Dmannitol, ra nose, 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 ssure 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 ow 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 lter 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.

Pathogenicity trial
The pathogenicity of Streptomyces strains was investigated using two cultivars known to be susceptible to ssure 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 lled halfway with the compost. One potato tuber was placed on top of the compost in each pot, after which the pots were lled 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 identi ed 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 ssure scab symptoms using a custom rating scale: Streptomyces species were re-isolated from potatoes with ssure scab symptoms. The isolates were puri ed and identi ed in order to ful l Koch's postulates. Data were analysed with SAS software (SAS Institute, Inc., 1999) to determine signi cant differences between treatments.

Sample collection and isolation
A total of 142 Streptomyces isolates were obtained from tubers with symptoms that resemble ssure scab. The symptomatic tubers were collected from all of the major potato production regions in South Africa.

Phylogenetic analyses
Newly generated DNA sequences from this study are available from GenBank with accession numbers MK934844-MK934992 and MK956209-MK956788. Isolates included in the phylogenetic analyses are listed in Table 1. Alignment of sequences for individual datasets yielded 1,367 bp (16S), 496 bp (atpD), 428 bp (gyrB), 504 bp (recA), 540 bp (rpoB) and 571 bp (trpB). The combined dataset of the 5 housekeeping genes yielded a dataset of 2,539 bp. The best substitution model for both 16S and combined datasets was GTR+I+G.
An initial ML tree consisting of all Streptomyces species and all ssure scab isolates were constructed (Fig. S1 supplementary material). The majority of the isolates (60%) obtained from ssure scab symptoms grouped into three clades. Clade 1 (17.6%) grouped with S. werraensis, clade 2 (17.6%) grouped close to S. pseudogriseolus and S. gancidicus, while clade 3 (24.6%) grouped close to S. aveolus. The other 49 isolates grouped into 37 different clades and these smaller groupings were not investigated further in the current study.
The tree topologies for the individual housekeeping genes were all congruent (data not presented) and showed concordance with the 16S rDNA tree topology (Fig. 2). The combined tree (Fig. 3) con rmed that the clade 1 isolates belong to species S. werraensis with a bootstrap support of 99%. Clade 2 isolates grouped close to S. pseudogriseolus, S. rubiginosus and S. gancidicus, and clade 3 isolates grouped close, but clearly distinct from S. aveolus with a bootstrap support of 99%.
The ANI and AAI with closely related Streptomyces are shown in Fig. 4; however, no genome sequence for S. werraensis was available. The values con rmed that clade 3 (isolates N26, FS66, FS70 and FS75) is a distinct species from S. aveolus with 84% ANI and 78% AAI. Clade 2 isolates (isolates N92 and FS94) are similar to S. pseudogriseolus and S. gancidicus which belong to one species as these isolates share 99% AAI and ANI.
The PAI commonly found in plant pathogenic Streptomyces species were not present in any of the six genomes. However, all six of the isolates contained analogues to the Staphylococcal pathogenicity islands (SaPI), while the clade 3 isolates also contained the Golgiassociated plant pathogenesis-related protein 1 gene.

Morphology and phenotypic analysis
Culture morphology on OA is shown in Fig. 5 a-c. Isolates belonging to clade 3 produced yellow diffusible pigments on all culture media (Fig. 5c), which made it possible to distinguish these cultures from clades 1 (Fig. 5a) and 2 (Fig. 5b). Some cultures in clade 2 produced a reddish-brown pigment on OMA. SEM revealed that clades 1 (Fig. 6a) and 3 (Fig. 6e) had mycelia in open loops (Retinaculiaperti), while clade 2 (Fig. 6c) formed simple spirals (Spirales). The spores surfaces of all three clades were spiny (Fig. 6b, d, f), with some spores in clade 1 exhibiting a warty surface (Fig. 6b). Although the culture morphology of clades 1 and 2 were similar, the distinct spirals of clade 2 and the profusion of single spores from clade 1 made it possible to distinguish between these clades.
Isolates from clades 1 and 3 were able to utilize L-arabinose, D-fructose, D-glucose, I-inositol, D-mannitol, ra nose, rhamnose, sucrose and D-xylose as sole carbon sources, however, utilization of sucrose, fructose and ra nose by isolates from clade 2 were doubtful (±). Isolates from all three clades were able to grow between pH 5.0-pH 11 and were tolerant to NaCl of up to 7.5%, but were inhibited by 10% NaCl. Growth studies were carried out on Streptomyces resiliuntiscabiei, sp. nov. and it was able to grow between 10 and 45 °C, with optimal growth between 30 and 35 °C. Streptomyces aveolus cannot utilize ra nose (Shirling and Gottlieb 1968), con rming that S. resiliuntiscabiei sp. nov. represents a distinct novel species.

Tuber slice assay
The three isolates causing the largest lesions from each of the three main phylogenetic clades were selected to use to test for pathogenicity in the glasshouse trial. These were isolates 057, FS29, FS97 (clade 1), FS33, FS94, N92 (clade 2), N26, FS70 and FS75 (clade 3).

Pathogenicity trial
Isolates from all three clades were able to cause symptoms similar to those seen in ssure scab (Fig. 7). The results from the glasshouse trial disease ratings are given in Fig. 8. In general, Innovator showed a higher scab index than Mondial, however it is not a statistically signi cant difference. Isolates FS97 (clade 1) and N26 (clade 3) resulted in a signi cant higher scab index on Innovator than on Mondial (p<0.05). However, there is large variation in the scab index within the same treatments.
Re-isolation and identi cation of pathogens from diseased tubers con rmed that the inoculated organisms were responsible for the disease symptoms observed.

Discussion
This is the rst comprehensive survey to identify the Streptomyces isolates associated with ssure scab in South Africa. The three main clades, comprising 60% of the isolates obtained from ssure scab symptoms from 2010-2018, were identi ed as S. werraensis, S. pseudogriseolus and S. resiliuntiscabiei sp. nov. Full genome sequences were generated for eight isolates selected from the three species. Glasshouse trials with the three species revealed that all three species are capable of causing ssure scab symptoms on potato cvs. Mondial and Innovator. The PAI genes present in the common scab pathogens, comprised of thaxtomin, tomatinase and nec1, are absent in most of the isolates, with only the nec1 gene being present in some isolates of S. resiliuntiscabiei (data not shown).
Horizontal gene transfer of the PAI has been proven to occur in Streptomyces species and to lead to the emergence of new pathogenic species (Bukhalid et al. 1998(Bukhalid et al. , 2002Kers et al. 2005;Zhang et al. 2016;Zhang et al. 2017). However, this PAI is absent in the ssure scab isolates, and this leads to the question of the mechanism of disease development. The genomes that were generated will be investigated further for indications on which genes may be responsible for initiating the cracks that are the main characteristic of the ssure scab symptoms.
The advent of DNA sequencing and more recently genome sequencing has removed the necessity of doing scores of phenotypic tests in the hope of nding morphological and phenotypic differences in order to describe a novel bacterial species. These tests are still useful in nding novel biochemical products produced by the ubiquitous Streptomyces genus, however, time and money spent by taxonomists will be greatly reduced without having to do these tests (Sutcliffe et al. 2012), while increasing the rate at which species can be described. As more full genomes become available, the use of the ANI and AAI statistics of these genomes will aid in making decisions on where to delimit species. The 16S rDNA as well as the combined phylogenetic trees in this study were inconclusive on the identities of clades 2 and 3. However, the ANI and AAI matrixes conclusively showed that clade 2 strains are members of S. pseudogriseolus, while clade 3 strains are not members of S. aveolus, but represent a novel species for which the name S. resiliuntiscabiei sp. nov. is proposed. The phenotypic, phylogenetic and genomic data support this proposal and the formal description of this new species follows below.
Description of Streptomyces resiliuntiscabiei sp. nov.
Streptomyces resiliuntiscabiei (resiliunt for the Latin word meaning "crack", describing the symptoms on the potato tubers).
Gram-stain positive, aerobic, non-motile, alkali tolerant and thermotolerant, with good growth of hyphae that are extensively branched with aerial hyphae that differentiate into open loops of spores (Retinaculiaperti). Good growth on ISP 3 and ISP 4, poor growth on ISP 2 and 5, and moderate growth on ISP 7. Colonies on ISP 3 are white initially, turning light grey after sporulation, surrounded with white margins. Diffusible yellow pigment discolours media. Isolates able to utilize L-arabinose, D-fructose, D-glucose, I-inositol, D-mannitol, ra nose, rhamnose, sucrose and D-xylose as sole carbon sources. Grow from pH 5.0-pH11. Grow well in the presence of 0-5% NaCl and can tolerate up to 7.5% NaCl, but were inhibited at concentrations of 10% and higher. Growth between 10 °C and 45 °C, with optimal growth between 30 °C and 35 °C.   Figure 1 Potato tubers with a. ssure scab, b. common scab and c. growth crack.

Figure 2
Midpoint rooted tree of Streptomyces species based on partial sequence of 16S rDNA gene region.    Symptoms on potato tubers after inoculation with Streptomyces species. Top row cv. Innovator and bottom row cv. Mondial.