Comparison between Pathogenic Streptomyces scabies Isolates of Common Scab Disease

Streptomyces scabies (Thaxter) causes destructive and serious damages to many vegetable field crops, including potato. Fourteen pure isolates were obtained from naturally diseased potato tubers showing symptoms of common scab disease, collected from different localities of Sohag governorate, Egypt. All tested isolates were identified as S. scabies (Stc) according to morphological and biochemical tests. Isolate Stc 10 exhibited the highest activity of polyphenoloxidase enzyme, followed by isolate Stc 11, while isolate Stc 2 produced the lowest activity of this enzyme. Concerning the peroxidase activity, the isolates varied in their production; Stc 11 exhibited the highest activity enzyme, followed by isolate Stc 2, whereas isolate Stc 10 produced the lowest activity of enzyme. In regard with Tyrosine Amonnia Lyase (TAL) activity, isolate Stc 2 exhibited the highest activity, followed by isolate Stc 10, whereas isolate Stc 11 exhibited the lowest activity. Agarose gel electrophoresis of the PCR amplification products revealed a band representing the expected 279 bp DNA fragment in each DNA extracted from the highly pathogenic isolates Stc 10 and 11. The results demonstrated that PCR amplification of the nec1 gene could be used as a reliable marker for detecting pathogenic Streptomyces isolates on potato tubers.


Introduction
There are many diseases that attack potato in the field crop among which common scab is one of the bacterial diseases that register high economic loses.Streptomyces scabiei (Thaxter) Lambert and Loria (syn.Streptomyces scabies) is the main causal agent of common scab, a widely-distributed disease that causes important economic wastes in potato production (Lambert and Loria, 1989;Goyer et al., 1996).Common scab symptoms consist of superficial to deep corky lesions on the tubers.Plant pathogenic Streptomyces spp.can survive in soil or plant debris for over a decade (Kritzman and Grinstein, 1991).
Streptomyces scabies strains were recently placed into three distinct genomic species based on DNA relatedness and 16S rDNA sequence, and assigned as either S. scabies, S. europaeiscabiei and S. stelliscabiei (Bukhalid et al., 2002).The nec1 gene encodes a secreted necrogenic protein with an uncharacterized plant cell target and was shown to confer a pathogenic phenotype on the nonpathogenic Streptomyces lividans (Bukhalid et al., 1998).The G + C content (54%) of nec1 is a typical relative to high G + C coding region, characteristic of Streptomyces strains, suggesting it was acquired through horizontal transfer from another toxin.The nec1 gene is also structurally and functionally conserved among unrelated pathogenic Streptomyces species and absent in nonpathogens from a wide geographic origin (Bukhalid et al., 2002;Park et al., 2003).In addition, there exists a strong correlation between thaxtomin A production and the presence of nec1 in pathogenic strains of S. scabies, S. acidiscabies and S. turgidiscabies (Bukhalid et al., 1998).However, the nec1 protein is not required for thaxtomin production and therefore it represents an independent virulence factor (Kers et al., 2005).
The hereby work aimed to isolate and identify the common scab pathogen, and to study the variation between the isolates of the pathogen by production of some enzymes, as evidence of the gene virulence.

Source of Streptomyces scabies, the pathogen of common scab disease
Isolates of Streptomyces scabies from diseased potato tubers harvested from Sohag Governorate, Egypt, were used.The pathogenicity of these isolates was previously tested by authors and they varied in their pathogenicity; from 14 isolates identified (Stc 1…14), in the hereby study, three isolates (Stc 2,10,11) with different degree in pathogenicity were used (Hosny et al., 2014).

Identification of pathogenic bacteria by morphological and physiological characteristics
The isolated bacterial isolates proved to be pathogenic and cause common scab to potato tubers; they were identified according to their morphological, cultural and physiological characteristics, as recommended by Krieg and Holt (1984), Schaad (1992), Holt et al. (1994).

Comparison between pathogenic isolates Certain enzyme production Peroxidase activity
One ml of ten days old bacterial culture was properly mixed in 2 ml sodium phosphate buffer 0.1 µ at pH 7.1.The homogenate was filtered through Whatman No.1 filter paper.The suspension was centrifuged at 6,000 rpm at 4 °C for 20 min and stored at -18 °C until use.
One tenth ml of the filtrate was added to 0.5 ml sodium phosphate buffer 0.1 µ at pH = 7.1, 0.1 ml H2O2 1% and 0.3 ml pyrogallol 0.05 µ.The mixture was completed to 3 ml using distilled water and color density was read in absorbance spectrophotometer model Miltonroy spectronic 601, at 425 nm every 30 seconds for 10 reads (Kochba et al., 1977).
Peroxidase activity was calculated as mg/g fw (fresh weight).
Phosphate buffer preparation was done with eight and nine tenth gram di-sodium hydrogen dissolved in 250 ml distilled water in a volumetric flask (solution A) and 3.9 gm sodium hydrogen phosphate was dissolved in 250 ml distilled water in another flask (solution B).Solution A was added to solution B to have pH 7.1.

Polyphenoloxidase activity
Enzyme samples were extracted as mentioned before in peroxidase activity extraction.One tenth extracted sample was added to 0.5 ml sodium phosphate buffer 0.1 ml at pH = 7 and 0.5 ml catechol 0.001 N. The mixture was completed to 3 ml using distilled water and color density was read in spectrophotometer (Miltonroy spectronic 601) at 495 nm every 30 seconds for 10 reads (Lisker et al., 1983).
Polyphenoloxidase activity was calculated as mg/g fw (fresh weight).

Tyrosine Amonnia Lyase (TAL) activity
Enzyme samples were extracted as mentioned before in peroxidase activity extraction.TAL activity was assayed by monitoring p-hydroxycinnamic acid formation at 310 nm using a double beam spectrophotometer (Uvikon, Kontron, Herts, UK) in 10 mM Tris buffer at 35 °C.The recommended pH was determined between the pH values 6 and 10.All reactions were performed in a total volume of 1 ml (Kyndt et al., 2002).
Tyrosine Amonnia Lyase activity was calculated as mg/g fw (fresh weight).

Gene virulence Extraction of microbial DNA from pure cultures
Following the growth of Streptomyces spp. on Yeast Malt Extract (YME) medium at 25 °C for 5-7 days, a 4 mm 2 plug of growth was used to inoculate 2 ml Tryptone 152 Soya Broth (TSB) containing 10% sucrose.Cultures were incubated at 25 °C at 100 rev min -1 for 2-5 days following which 1.5 ml of culture was removed and centrifuged at 11,550 g for 4 min to pellet cells.Supernatant was discarded and the pellet was re-suspended in 0.5 ml sterile quarter strength Ringer's solution (QSR) (Oxoid UK catalog No. BR52) and vortexed.Suspensions were centrifuged at 11,550 g for 2 min to pellet cells and the QSR solution was discarded.The cell pellet was re-suspended in 1.0 ml CTAB extraction buffer (2% CTAB, 1.5 mol l -1 NaCl, 0.2 mol l -1 Tris, 25 mmol l-1EDTA; pH 8.0) and the suspension transferred to a 2 ml Eppendorf tubes containing 0.1-0.2g of 1.0 mm glass beads.
Cells were lyses by physical disruption in a Mini-Bead Beater-8 (Bio-Spec Products, Bartlesville, OK, USA) at a medium setting for 1 min.Tubes were centrifuged (11,550 g for 2 min) to pellet cell debris, and the supernatant was transferred to new 2 ml Eppendorf tubes and then extracted with an equal volume of chloroform, mixed and recentrifuged (11,550 g for 5 min).DNA in the aqueous phase was precipitated with 0.3 mol l -1 sodium acetate (pH 5.2) and an equal volume of isopropanol, for 1 h at room temperature.The DNA was pelleted by centrifugation (11,550 g for 5 min), washed in 70% ethanol, re-pelleted, re-suspended in 100 µl TE (10 mmol l -1 Tris-HCl and 1 mmol l -1 EDTA, pH 8.0) and purified by Sephadex G-75 spin column chromatography using the method of Cullen and Hirsch (1998).
DNA was quantified using a TKO 100 Mini-Fluorometer (Hoefer Scientific Instruments, San Francisco, CA, USA) following the manufacturer's instructions and the quality was checked by agarose gel electrophoresis.

Design of nec1-specific PCR primers
A set of 20-mer primers was designed to target the coding sequence of the virulence nec1 gene of S. scabies.The expected amplicon size amplified by the (Nec-NF1) PCR primer was 279 bp (Table 1).The conventional PCR as previously designed by Bukhalid et al. (1998) was followed.
PCR methods and reagents PCR amplification of DNA samples was based on an initial denaturation at 95 °C (2 min), followed by 35 cycles of denaturation at 95 °C for 45 s, annealing at 65 °C for 1 min, extension at 72 °C for 90 s and a final elongation at 72 °C for 5 min in a reaction volume of 25 µl using a GeneAmp PCR System 9600 thermal cycler (Applied Biosystems).PCR products were analyzed by electrophoresis on 2% agarose gels (Sambrook et al., 1989).

Statistical analysis
Analysis of variance (ANOVA) was carried out using MSTAT-C program.The Least Significant Difference (L.S.D) at P ≤ 0.05 was applied to detect differences among treatments (Gomez and Gomez, 1984).

Identification of the causal pathogens
Identification of the isolated pathogenic bacteria was carried out using the morphological and physiological characteristics.
Data presented in Table 2 indicated that all tested isolates were of filamentous (or spiral) shape, non-motile, sporing, gram positive, gelatin liquefaction positive, starch hydrolysis positive, urease negative, catalase test positive, esculin hydrolysis positive antibiotic positive, no growth at both 4 °C and 40 °C, H2S production negative, levan production negative, methyl-red test negative, phenyl alanine deminase test negative, casein hydrolysis positive.
Accordingly, data in Table 3 indicated that all tested isolates produced acid and did not produce gas from glucose, fructose, sucrose, lactose, maltose, manitol, dextrin and anhydrous dextrose.
On the basis of morphological, cultural, physiological and pathological characteristics of the isolated bacteria, it was concluded that all tested isolates could be identified as Streptomyces scabies.

Comparison between pathogenic isolates Certain enzymes production
This experiment was conducted to study the variation of enzyme production, Peroxidase (PO), Polyphenoloxidase (PPO) and Tyrosine amonnia lyase (TAL) by three isolates: isolates Stc 11 and 10, the highly pathogenic isolates, compared with isolate No. 2 as the lowest pathogenic isolate.
Results of the current experiment are represented in Table 4 and indicated that isolate Stc 11 exhibited the highest activity of peroxidase enzyme, followed by isolate Stc 2, whereas isolates No. 10 exhibited the lowest activity.
Results also indicated that isolate Stc 10 exhibited the highest activity of polyphenoloxidase enzyme, followed by isolate Stc 11, whereas isolate Stc 2 showed the lowest activity of enzyme.Concerning TAL activity, isolate Stc 2 exhibited the highest activity followed by isolate Stc 10, whereas isolate Stc 11 exhibited the lowest activity.

Pathogenic isolates comparison by PCR technique
Streptomyces scabies isolates Stc 10 and 11 which had been previously confirmed as highly pathogenic isolates, and isolate Stc 2 which had been previously confirmed as low-pathogenic one, were selected and checked by PCR method.Agarose gel electrophoresis of the PCR amplification products revealed a band representing the expected 279 bp DNA fragment in each DNA extracted from the highly pathogenic isolates Stc 10 and 11.

Discussion
Results reported herein indicated that the fourteen bacterial isolates obtained from naturally diseased potato tubers collected from different localities of Sohag governorate proved to be pathogenic and able to infect potato tubers, causing common scab symptoms.They were identified as Streptomyces scabies.These results are in agreement with those noted by several researchers who reported that S. scabies was responsible for common scab of potato (Lambert and Loria, 1989;Goyer and Beaulieu, 1997;Bencheikh and Setti, 2007;Manome et al., 2008).
The experiment was conducted to determine the role of certain enzymes in the virulence of Streptomyces scabies isolates.The activity of Peroxidase (PO), Polyphenoloxidase (PPO) and Tyrosine Amonnia Lyase (TAL) enzymes in the highest pathogenic isolates Stc (10 and 11) compared with the lowest disease index isolate Stc 2.
Results of this experiment indicated that the tested isolates varied in their production of the analyzed enzymes.Isolate Stc 11 exhibited the highest activity of peroxidase enzyme, followed by isolate Stc 2, whereas isolates Stc 10 exhibited the lowest activity.Also, isolate Stc 10 exhibited the highest activity of polyphenoloxidase enzyme, followed by isolate Stc 11, then isolate Stc 2. Finally, isolate Stc 2 exhibited the highest activity of TAL enzyme, followed by isolate Stc 10, whereas isolate Stc 11 exhibited the lowest activity.These results may indicate that there was no correlation between the activity of these enzymes and the severity of common scab disease attack.Such results are in disagreement with those reported by Shihata (1974) who mentioned that highly pathogenic isolates were less active in B-glucosidase, Polyphenoloxidase and Peroxidase enzymes, followed by moderately pathogenic isolates and finally weakly pathogenic isolates.
PCR techniques were developed in the current study for the specific detection of the virulence gene nec1 of pathogenic Streptomyces isolates on potato tuber samples.This assay was based on the design of Nec1 (279 bp) primer by using PCR assays which were able to distinguish pathogenic Streptomyces species nec1 from low pathogenic ones.
Data hereby obtained demonstrated a clear correlation between pathogenicity of Streptomyces isolates and the detection of the nec1 gene by conventional PCR.These results were similar with thise reported by Bukhalid et al. (2002).The low-pathogenic isolate Stc 2 of Streptomyces lacked the nec1 gene.
The previous work carried out by Bukhalid et al. (2002) reported that the sequence of nec1 was identical in all pathogenic strains, irrespective of their geographic origin, isolation host, or level of DNA relatedness to the Streptomyces scabies type strain, thus being a eliable marker for detecting pathogenic Streptomyces isolates on potato tubers.Such an assay could also have practical implications as a rapid substitute for pathogenicity tests involving the use of potato tubers (Schaad, 1988).

Table 1 .
PCR primer designed for specific detection of the nec1 gene of pathogenic Streptomyces scabies

Table 2 .
Morphological and physiological characteristics of the isolated pathogenic isolates of Streptomyces scabies

Table 3 .
Fermentation of carbon compounds by the isolated pathogenic isolates of Streptomyces scabies

Table 4 .
Peroxidase, Polyphenoloxidase and Tyrosine amonnia lyase activity in three S. scabies isolates Different letters indicate significant differences among treatments within the same column according to L.S.D (Least Significant Difference test 5%).