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2nd International Conference On Biotechnology Applications In Agriculture (ICBAA), Benha University, Moshtohor and Hurghada, 8-12,April 2014, Egypt

DNA-Polymorphism among some Streptomyces scabies isolates causing potato common scab disease using RAPD-PCR technique

Abdou M.M. Mahdy*; Ali. E. Tawfik **;F. G. Mohamed, *; R.N.Fawzy* and Omnia. A.Abd- El –Hafez**

* Agric Botany Dept.,Plant Pathology Branch, Fac. Agric., Benha Univ.-Egypt.** Plant Pathology, Research Institute, Agricultural ResearchCentre, Giza, Egypt.

AbstractFifty seven Streptomyces isolates were isolated from scabbed tubers and soil of infected potato fields. The

isolates were classified based on color of colonies, spores and diffusible melanin production and sporphore morphology. They synthesized pigment when grown on MSSA, YME and OMA-T media and they seemed to have utilized different ISP carbon sources. Twenty two isolates of Streptomyces were selected to use in the further trials. These twenty two isolates differed in their utilization abilities to different ISP carbon sources and the most appropriate chemicals for growth of isolates were glucose followed by raffinose, mannitol and arabinose, respectively. Also, they were Gram positive and had positive infection of roots and shoots of radish seedlings. Variation in aggressiveness was observed among isolates when using mini-tubers assay in pathogenicity. Genetic diversity among thirteen Streptomyces scabies isolates were studied using three primers i.e., 1, 6 (10 mer) and P (15 mer). Primer 1 was the best in revealing the clear similarity and diversity among the tested Streptomyces isolates.

Key words: Common scab, Streptomyces scabies, Biochemical characteristics,RAPD-PCR.

Introduction

Potato common scab occurs in all potato growing areas of the world (Hooker, 1981; Loriaet al., 1997 and Kreuzeet al., 1999). Potato scab has limited effect on tuber yield but can greatly affect tuber quality and therefore, severely reduces the marketability of table stock and tuber seeds this is due to the external symptoms on diseased tubers. Common scab of potato characterized by deep-or shallow- pitted lesions on tubers which are caused by Gram-positive filamentous bacteria belonging to the genus Streptomyces which was first described by Thaxter (1891).More than 400 Streptomyces species were described, but limited number of species is pathogenic to plant. Isolation from plant tissues or soil often yield a high percentage of saprophytic strains. This is very important to check for pathogenicity before any further characterization. The known species of plant pathogenic streptomyces are differentiated on basis of color of sporulation colonies in YME(yeast malt extract) medium, spore chain type ,production of melanin and utilization of all sugars as recommended by the International Streptomyces Project (ISP) .The known plant pathogenic Streptomyces include 11 species Lambert and Loria (1989). Wen et al. (2012) isolated the streptomycete strain SCY114 from a soil sample of Xuchang in Henan province, China. It possessed smooth grey spores born in rectiflexible and spiral chains and was capable of using all of the International Streptomyces Project sugars.

Bouchek- Mechicheet al.(2000); Loriaet al. (2001) and Lenhtonen et al. (2004)developeda time-saving and cost-effective polymerase chain reaction

(PCR)-based method for species-specific detection of the scab pathogens (Streptomyces scabies and S. turgidiscabies) prevalent in potato (Solanum tuberosum) in Northern Scandinavia where species specificity of primers were verified using a collection of previously characterized Streptomyces strains isolated from potato scab lesions in Finland and Sweden.Malkawi et al. (1999)used Random Amplified Polymorphic DNA (RAPD) for identification and assessment of genetic diversity between isolates of Streptomyces. Genomic DNA from 22 Streptomyces isolates were amplified using three different 10-mer primers. Different DNA finger printing patterns were obtained for all the isolates.

The present study was designed to identify and characterize of the pathogenic Streptomyces strains which cause potato common scab in Egypt.

Materials and Methods

1- Source of common scab pathogen samples:Samples of diseased potato tubers with visible

lesions on tubers were collected freshly from different localities of potato production areas in Menuofia, Gharbia, Beheira, Ismailia and Qalyubia Governorates, some markets and pack-houses in Egypt. Sandy and clay soil samples were also collected at 0- 15cm depth of grown potatoes in the field and used for isolation of potato common scab pathogens.

2-Isolation from scabbed tubers:The causal agents of potato common scab disease

were isolated from apparently infected tubers as described by Loria and Davis (1989), onto YME,

Plant pathology bio-techniques, 15-24 15

Polymorphism among some Streptomyces scabies…………..

OMA or OMA-T. The plates were incubated at 28°C for 7 days, then the appeared colonies with powdery texture were selected and their colors were characterized to obtain pure culture for use in the further trails.

3-Isolation from soil samples:Soil samples were placed in a sterile plates and

left to dry overnight at room temperature to reduce the number of unwanted bacteria. Then the samples were analyzed as described by Keinth and Loria (1989). Serial of ten-fold dilutions were made in sterile distilled water (SDW) and triplicate samples of 0.1 mL of undiluted, 10² and 104 dilutions were separately spread evenly on Petri plates containing oat meal agar (OMA) medium amended with tyrosin (5g/L), (OMA-T) penicillin (1mg/L),polymixin B sulfate (10mg/L) and cycloheximide (100 mg/L) .

The plates were then incubated at 28°C, for 7 days then the appeared colonies were characterized for S. scabies (melanin production and mycelium with gray color on OMA-T).

4-Pathogenicity tests :a-Radish seedling assay :

According to Leiner et al. (1996) the effect of Streptomyces isolates on radish seedling was evaluated. Seeds were surface disinfested by immersing them for 3 min. in a solution containing 0.5 % sodium hypochlorite and 0.1% Tween 20. The seeds then were rinsed three times in SDW and placed onto 1.5% water agar (WA) plates and then incubated at room temperature for 24 hrs. to allow them to germinate. Germinated seeds individually placed in glass tubes (25 mm diameter) containing 10ml of 1% WA. Then, 300 µL of tested culture suspension were added to each tube to inoculate seeds. Three replicates of each isolate were used. The inoculated tubes were kept at room temperature and examined daily. Pathogenic isolate causing brown to black lesions and stunting of the seedlings were selected. Radial swelling was sometimes visible. SDW treatment was used as a negative control and a known pathogenic Streptomyces strain was used as a positive control.

b- Minitubers assay:The leaf bud cuttings method described by Lauer

(1977) was used to test the Streptomyces isolates for their pathogenicity. Susceptible potato plants (Spunta) were grown under greenhouse conditions in 15 cm plastic pots containing sterile substrate (2/3 peat and 1/3 sand). Leaf bud cuttings (leaf with an axillary bud a short – stem section) were cut from the potato plant and immediately transferred to pots filled with water-saturated sterile sand. The stem cuttings were planted deep enough to cover the axillary buds. The sand was kept moist. After 15 days, the mini-tubers began to form from the axillary buds and ready for inoculation. Each mini-tuber was

inoculated by adding 500µL suspension each one of the Streptomyces tested isolates. The inoculated tubers were again covered with sand and the soil moisture was maintained to keep the cuttings alive. Non-inoculated mini-tubers were used as a negative control to be sure that the inoculum was not coming from the soil.

Potato common scab disease index:The disease index (DI) was determined by

counting number of lesions in 1 cm² of tuber surface in different treatments according to the scab proposed by Shihata (1974) using the following category.

DI% = 0A+1B+2C+3D+4E+5F× 100 5T

Where: 0 = no scab. 1 = trace – 10% tuber surface is scabbed. 2 = 11 – 20 % tuber surface is scabbed. 3 = 21 –30 % tuber surface is scabbed. 4 = 31 – 40 % tuber surface is scabbed. 5 = more than 40 % tuber surface is scabbed.

A, B, C, D, E and F are the number of tubers corresponding to the numerical grades respectively.

T = is the total number of tubers, i.e. T = A+ B+ C+ D+ E+ F.

5-Morphological, physiological and biochemical characteristics:

Streptomyces isolates were identified according to Bergy's manual of systematic bacteriology (Krieg et al., 1994).

5- 1-Morphological characteristics :

a- Aerial mass color:Each isolate was cultured for 14 days in the dark

at 30°C on four different media, YME, oat meal (OA), inorganic sodium salts agar (ISSA) and glycerol asparagine agar (GAA).The colour of the mycelium and spores was recorded as white, white-yellow, red, brown or gray. The isolates were classified according to Tresner and Backus (1963).

b- Reverse pigmentation (colony color):Grouping was based on the production of

characteristic diffusible pigments in the reverse of colonies on YME, OA, ISSA and GAA media.c- Spore chain morphology:

To observe sporulation, the isolates were inoculated and grown around microscope slip obliquely inserted in YME plates. The cover slips were removed after sporulation and then the adhering mycelium and spore chains were observed under a light microscope at 100x magnification and described as straight, flexuous or spiral.

2-Physiological characteristics:

Plant pathology bio-techniques 16


a- Carbon utilization:The test was described by the International

Streptomyces Project (ISP) (Shirling and Gottieb, 1966). Sugar utilization was recorded as presence or absence of growth on two Petri dishes of basel mineral salts agar amended with 1% of the following sugars: (D– fructose, D- glucose, D- mannitol, D- raffinose, D- xylose, L-arabinose, cellibiose, inositol , sucrose).

b-Melanin production :Peptone-yeast extract agar (PYA) and oat meal

amended with tyrosine (OMA-T) slants were inoculated in triplicates with each Streptomyces isolate. Un-inoculated PYA and OMA-T slants were inoculated as references. The slants were incubated at 28°C in the dark and observed after 2 and 4 days. Isolates which produced greenish brown to brownish black pigment on both media were considered as melanin producers.

6- RAPD-PCR technique in identification and characterization of Streptomyces isolates:- Culturing of Streptomyces spp. strains:

Seven days old culture of Streptomyces strains were grown onto OMA (oat meal agar) at 28°C then the mycelium and spores were scraped from the surface of the medium. Then, they were placed in test tubes each containing 5 ml of tryptone/yeast extract broth medium. The tubes were shaken (to homogenize the samples) in a rotary shaker at 100 rpm for 72 h at 30⁰C.

DNA extraction and amplification DNA was isolated from 50 mg of each isolate

using a Qiagen; Kit for DNA extraction. The extracted DNA was dissolved in 100 uL of elution buffer. The concentration and purity of the obtained DNA was determined using “Gen qunta” System-Pharmacia BioTech. The purity of the DNA for all samples must be between 90-97% and the ratio between 1.7 – 1.8. Concentrations were adjusted at 6 ng/ uL for all samples using TE buffer pH. 8.0.

Thirty ng of extracted DNA and 0.40 µM of the three tested 1, 2 and P primers were used for amplification reaction. The PCR mixture contained PCR beads tablet (manufactured by Amessham Pharmacia Biotch), containing all necessary reagents except the primers and the DNA which add to the tablet. The sequences of the used primers 1 and 6 (10 mer) and primer P (15 mer) are shown in Table1

Table 1. Sequences of the three primers used in RAPD-PCR.

Base sequencePrimer Number

5-GGTGCGGGAA-315-CCC GTC AGCA-365-TCT GAC CCC CGT CAC-3

P

The total volume was completed to 25 µL using sterile distilled water. The amplification protocol was carried out as follows using PCR unit II biometra. For (1, 2 and P) primers: Denaturation at 95 ˚C for 5 min.45 cycles each consists of the following steps:1)-Denaturation at 95 ˚C for 1 min.2)-Annealing at 40 ˚C for 1 min.3)-Extension at 72 ˚C for 2 min.Final extension at 72 ˚C for 5 min.Hold at 4 ˚C7 µL of 6 X tracking buffer (manufactured by Qiagen Kit) were added to 25 µL of the amplification product.The amplified DNA of all samples were electrophoresed (15µL, using an electrophoresis unit WIDE mini-sub-cell GT Bio-RAD) on 2% agarose containing ethedium bromide (0.5 μg/mL), at 75 constant volt, and determined with UV transilluminator .

Gel analysis:The DNA were scanned for band Rf using gel

documentation system [AAB Advanced American Biotechnology 1166 E. Valencia Dr. Unit 6 C, Fullerton CA 92631]. The different M.W. of bands were determined against PCR marker(Resco K180) 100 bp DNA by un-weighted pair-group method based on arithmetic mean (UPGMA)

Results

1- Phenotype characterization of isolated common scab strains from scabbed tubers

Fifty seven isolates were obtained from naturally infected potato tubers and various soils, as well as, from imported tubers.

The cultural and morphological characteristic of the isolates on the media (MSSA, YME, OMA-T) are presented in Table 2. Isolates were classified on basis of colour of colonies and spores (as:grey, white, yellow, red and brown), and diffusible melanin production, sporophore morphology (as:spiral, flexuous and branched)

Pathogenicity tests:Pathogenicity tests of the twenty two isolates

were performed on radish seedlings and potato mini tubers of cultivar Spunta. Data presented in Table 3 show that all isolates had positive infection differentiation of roots and shoots of radish seedlings which reduce the length of shoots and roots compared with the control.

Variation in aggressiveness was observed between isolates when using the mini-tuber assay in pathogenicity. No clear lesions were observed with isolates SS-16, T-32, T-41 and T-44. Lesions of scab on progeny tubers appeared with all of the remaining isolates with different degrees due to their high aggressiveness. Pathogen reisolated from inoculated



progeny tubers had spore colors and morphologies identical to those of the original pathogen.

Table 2. Phenotypic characterization of fifty seven isolates of Streptomyces scabies causing common scab disease which isolated from scabbed tubers and soil samples.

Isolate No. Locality Source of isolationSpore chain

type (medium)*

Aerial colourof colony

(medium)

Pigments production (medium)

CS-1CS-2CS-3CS-4CS-5CS-6CS-7CS-8CS-9CS-10CS-11CS-12SS-13SS-14SS-15SS-16SS-17SS-18SS-19SS-20SS-21SS-22SS-23SS-24SS-25SS-26SS-27T-28T-29T-30T-31T-32T-33T-34T-35T-36T-37T-38T-39T-40T-41T-42T-43T-44T-45T-46T-47T-48T-49T-50T-51CS-52CS-53CS-54CS-55CS-56CS-57

MenufiaMenufiaMenufiaMenufiaMenufiaMenufiaMenufiaMenufiaMenufiaMenufiaMenufiaMenufiaDena 4Dena 7Dena 4Dena 7Dena 7Dena 2Dena 6Dena 2Dena 2Dena 7Dena 4Dena 4Dena 4Dena 6Dena 2ImportationBadr regionMarket (L)ImportationDemiatt Prov.Demiatt Prov.Demiatt Prov.Demiatt Prov.ImportationBadr regionBadr regionBadr regionBadr regionImportationImportationImportationImportationMarket (L)ImportationIsmailia Prov.Market (L)ImportationImportationImporationMoshtohr

Clay soil (pH7.8)Clay soil (pH7.8)Clay soil (pH7.8)Clay soil (pH7.8)Clay soil (pH7.8)Clay soil (pH7.8)Clay soil (pH7.8)Clay soil (pH7.8)Clay soil (pH7.8)Clay soil (pH7.8)Sandy soil (pH8.1)Sandy soil (pH8.1)Sandy soil (pH8.1)Sandy soil (pH8.1)Sandy soil (pH8.1)Sandy soil (pH8.1)Sandy soil (pH8.1)Sandy soil (pH8.1)Sandy soil (pH8.1)Sandy soil (pH8.1)Sandy soil (pH8.1)Sandy soil (pH8.1)Sandy soil (pH8.1)Sandy soil (pH8.1)Sandy soil (pH8.1)Sandy soil (pH8.1)Sandy soil (pH8.1)Russet spotsRusset spotsRaised spotsRaised spotsNetted spotsRaised sportRusset spotsNetted spotsRaised spotsRusset spotsRusset spotsNetted spotsNettle spotsRaised spotsRaised spotsRaised spotsRusset spotsRaised spotsRusset spotsRaised spotsNetted spotsNetted spotsPitted spotsRusset spotsClay soil (pH8.3)Clay soil (pH8.3)Clay soil (pH8.3)Clay soil (pH8.3)Clay soil (pH8.3)Clay soil (pH8.3)

SpiralSpiralSpiral

BranchedBranchedStraightFlexuousFlexuous

SpiralSpiralSpiral

FlexuousSpiralSpiralSpiralSpiralSpiralspiral

FlexuousFlexuousflexuousBranchedBranched

SpiralFlexuousFlexuousFlexuous

SpiralSpiralSpiral

BranchedBranchedBranchedBranchedBranched

SpiralFlexuousFlexuousFlexuous

SpiralSpiral

StraightStraightStraightFlexuousFlexuousFlexuous

SpiralSpiralSpiralSpiral

FlexuousFlexuousFlexuous

SpiralSpiralSpiral

WhiteGrey

Deep blueDeep blueDeep blue

OrangeGrey

YellowWhiteGreyWhiteBrownGreyWhiteBrownWhiteWhiteWhiteGreyGreyWhiteWhiteWhiteWhiteBrownBrownYellowYellowWhiteWhite

Deep redWhiteWhiteWhiteYellowGreyWhiteGrey

YellowYellowGrey

YellowGreyGrey

YellowYellowBrownWhiteYellowGreyGreyGrey

Deep redWhiteWhiteYellow

red

MelaninMelaninMelaninBlackBlackRedRed

YellowYellowMelaninMelaninBlack

--

Melanin---

BlackBlackBlackRedRedRed

----

MelaninMelaninMelaninMelaninMelaninBlackBlack

---

RedRed

MelaninMelanin

---

BlackBlackBlackRedRedRed

MelaninMelaninBlackBlackBlackBlack

* Growth medium:1. Modified salt starch agar (MSSA).



2. Yeast malt extract (YME).3. Oat meal agar + tyrosine (OMA-T).

Table 3.Pathogenicity tests for 22 pathogenic isolates of Streptomyces scabies.

Isolate No. Locality Sourceof isolation Type of ScabPathogenicity Tests

Radish Seedling Minituber Assay

SS-16 Dena 7 Sandy soil Superficial + +SS-22 Dena 7 Sandy soil Superficial + +SS-23 Dena 4 Sandy soil Superficial + +SS-27 Dena 2 Sandy soil Superficial + +T-28 Importation tuber Russet spot + +T-29 Badr region tuber Russet spot + +T-30 Market (L) tuber Raised spot + +T-31 Importation tuber Raised spot + +T-32 Demiatt Prov. tuber Netted spot + NDT-33 Demiatt Prov. tuber Raised spot + +T-34 Demiatt Prov. tuber Russet spot + +T-36 Importation tuber Raised spot + +T-37 Badr region tuber Russet spot + +T-38 Badr region tuber Russet spot + +T-39 Badr region tuber Netted spot + +T-40 Badr region tuber Netted spot + +T-41 Importation tuber Raised spot + NDT-42 Importation tuber Raised spot + +T-44 Importation tuber Raised spot + NDT-46 Importation tuber Russet spot + +T-47 Ismailia tuber Raised spot + +T-49 Importation tuber Netted spot + +

+ : Positive reactionND : Not determined

Utilization of different ISP carbon source by isolated organisms: Table 4 shows results of tested twenty two isolates of Streptomyces to utilize the different carbon sources. All isolates were differed in their utilization abilities with regard to raffinose, mannitol, inositol and cellibiose. Source of carbon like fructose was not utilizedby some isolates (T-31,T-32,T-33,T-34,T-36) which were able to utilize other sugars. However, the most appropriate chemicals for growth of isolates were glucose, raffinose, mannitol, and arabinose.

Biochemical and physiological characters of isolated organisms: Data in Table 5 indicate that all twenty two isolates were G+ve, catalase positive reaction, aerobic, starch hydrolysis, gelatin liquefaction, and reduced nitrate to nitrite.All isolates gave negative V.P. and M.R. tests and also negative indole formation.

Random amplified polymorphic DNA (RAPD)Total DNA from thirteen different Streptomyces

scabies isolates were extracted and three primers were selected for initial screening by RAPD fingerprinting. All primers produced distinct fingerprints. Two of them consisted of 10 mere (primer 1&6). The other consisted of 15 mere. Primer 1 produced the most distinct pattern, with up to 13 bands had different size in each isolate.

Results using primer 1: The 13 isolates into 4 subgroups. (Fig 1and2)The first subgroup include isolates n 1, 2, 4 and 5. Isolates 1and2 were imported from Europe. Isolates 4and5 were isolated from a sand soil in Alexandria governorate, with 96.38% similarity between isolates 2and4 similar with isolate 5 with 91.79% and 85.51% with isolate 1.

Fig.1. 1.5% Agarose gel electrophoresis showing RAPD amplification products of Streptomyces isolates using primer 1. M: 100bp DNA ladder marker.

M 1 2 3 4 5 6 7 8 9 10 11 12 13 bp

3000 2000 1500 1200 1000 900 700 500 400 300 200 100

Lane M: the marker Lane 7: isolate T-29 Beheira .Lane 1: isolate T-51 Europe. Lane 8: isolate T-30 Benha.Lane 2: isolate T-50 Europe. Lane 9: isolate T-40 Beheira.Lane 3: isolate SS-22 Alexandria. Lane 10: isolate T-45 Giza.Lane 4: isolate SS-23 Alexandria. Lane 11: isolate T-48 Giza.Lane 5: isolate SS-26 Alexandria. Lane12: isolate CS-54 Moshtohr.Lane 6: isolate T-28 Europe. Lane13: isolate T-49 Europe.



Table 4. Reaction of twenty two pathogenic isolates by utilization test of sugars which having International Streptomyces Project (ISP).

Isolate No

Ara

bino

se

sucr

ose

Fruc

tose

Xyl

ose

Raf

finos

e

Man

itol Positive

control G

lucose

Negative

control Inos

itol

Cel

libio

se

SS-16 ++ + + + ++ + + - ++ +SS-22 + + + + ++ + + - + ++SS-23 ++ ++ ++ + ++ ++ + - ++ ++SS-27 ++ ++ ++ + ++ + + - ++ ++T-28 + + + + ++ ++ + - + ++T-29 + + - ± ++ + + - ++ +T-30 + + ± + ± + + - + ++T-31 + ++ - + ± + + - + ++T-32 + ++ - ± ++ + + - ++ ++T-33 ++ ++ - ++ ++ ++ + - ++ ++T-34 ++ + - - ++ ++ + - ++ ++T-36 + + - + + + + - + +T-37 ++ ++ + ++ + ++ + - ++ +T-38 ++ ++ + ++ + ++ + - ++ +T-39 + + ± ++ + ++ + - + +T-40 + + ± ± + + + - + +T-41 + + - + + + + - + +T-42 + + + ++ ++ ++ + - + ++T-44 + + ++ + ++ + + - + +T-46 ++ ++ + + + ++ + - + ++T-47 + + - + ++ ++ + - + ++T-49 ++ + ++ ++ ++ ++ + - ++ ++

++ Strong utilization of sugar .+ Moderate utilization of sugar .±Weak utilization of sugar .- Negative utilization of sugar .

Table 5 .Biochemical and physiological tests of Streptomyces

Indo

l fo

rmat

ion

Tyr

osin

-an

se

M.R

.te

st

V.P

.te

st

Nitr

ate

redu

ctio

n

Gel

atin

liq

uific

atio

n

Star

ch

hydr

olas

e

Aer

obio

sis

Cat

alas

e ac

tivity

Gra

m

reac

tion

Isol

ate

No.

-+--+++aerobic++SS-16-+--+++ aerobic++SS-22-+--+++aerobic++SS-23-+--+++aerobic++SS-27-+--+++aerobic++T-28-+--+++aerobic++T-29-+--+++aerobic++T-30-+--+++aerobic++T-31-+--+++aerobic++T-32-+--+++aerobic++T-33-+--+++aerobic++T-34-+--+++aerobic++T-36-+--+++aerobic++T-37-+--+++aerobic++T-38-+--+++aerobic++T-39-+--+++aerobic++T-40-+--+++aerobic++T-41-+--+++aerobic++T-42-+--+++aerobic++T-43-+--+++aerobic++T-44-+--+++aerobic++T-46-+--+++aerobic++T-47-+--+++ aerobic++T-49

isolates from scabbed tubers and soil sample .Polymerase chain reaction (PCR)



Fig2. The cluster analysis of the thirteen Streptomyces isolates amplified using primer 1

The second subgroup includes isolates 8, 9, 11 and 13. Isolates 8 and11 were local isolates isolated from the market. And the isolate 9 was local isolate from Beheira governorate. Isolate 13 was imported and seemed of similarity ranging from 86.25% to 95.51%. The third subgroup showed 96.47%similarity between isolates 3and 10; isolated from Alexandria and Giza governorate respectively and were similar to isolate7(isolated from Beheira governorate) with 79.25%. The fourth subgroup consisted of isolate 6 (from Europe) and isolate 12 (from Qalybia governorate) with 66.79 % similarity.

When primer 6 was used another classification was shown. There were thirteen isolates in six subgroups. As shown in Figs 3and4. Isolates no 1 and 11 were separated into separate groups from the rest of the isolates and each one was put in a separate group. The remaining isolates were classified into four subgroups. The first subgroup contained isolates 2 and 3 with 86.49% similarity, isolates 9 and12 with 94.19% similarity. Isolates 10 and 13 showed 90.36% similarity. Isolate 5 was of 73% similarity with isolate 7 in the second subgroup. The third subgroup included isolates 4 and 8 with 66.26% similarity. The similarity between all isolates was 21%.

Fig.3. 1.5% Agarose gel electrophoresis showing RAPD amplification products of Streptomyces isolates using primer 6. M: 100bp DNA ladder marker.

Fig.4. Cluster analysis of the thirteen Streptomyces isolates, amplified using primer p.

In order to detect markers for all these isolates a 15 bp primer was used with all isolates which represent new classification, with low similarity between isolates, as shown in Figs 5 and 6. Isolates 1and 5 have 55.81 % similarity. The remaining isolates were classified into three subgroups. The first subgroup has isolates 12, 10, 4and 3. The second has isolates 5, 7, 8, 9 and 13. The third contains isolates no. 5 and11. And all isolates has low similarity degree 18%.

Fig.5. 1.5% Agarose gel electrophoresis showing RAPD amplification products of Streptomyces isolates using primer P. M: 100bp DNA ladder marker.

Fig.6. Cluster analysis of the thirteen Streptomyces isolates, amplified using primer P.



Discussion

The obtained results of isolation trials yielded twenty two pathogenic isolates of Streptomyces scabies with different degree of virulence. Such findings do not coincide with those reported by Millard (1926) who reported advanced certain evidence that pathogenic species of Streptomyces scabies show their virulence in soil after prolonged existence as saprophytes. This also may indicate that avirulent isolates isolated from the infested soils and that causing the infections in the field are usually originated from infections already present on the mother tubers used for planting. Further studies are needed to find out whether or not the virulent retains of Streptomyces scabies left over in soil after harvesting diseased crops may lose their virulence. Twenty two isolates were identified according to morphological, physiological and biochemical characteristics advocated by the ISP (International Streptomyces Project) Shirling and Gottlieb (1966) and Bergy's manual. The pathogenicity tests showed that the Streptomyces isolates caused reduction of the length of radish seedlings and variable radial swelling were observed due to the virulence of the isolates. These results are in agreement with Liu et al., (2004) and indicate that the pathogenic isolates of Streptomyces scabies induced variable phenotypes of scab on potato mini-tubers which ranged from small lesions to necrotic area of scab symptoms. Such data are in harmony with El-Sheikh, (2010). It can be concluded that the presence of thaxtomine in the tested isolates will induce stunting of the seedlings and radial swelling. SDW is used as negative control. This finding are in agreement with Lawerence et al., (1990) and King et al., (1992) who identified the phytotoxins from Streptomyces isolates as thaxtomine which was involved in infection of potato with Streptomyces scabies. It is important to consider evaluation of pathogenicity assay by the effects of thaxtomine as similar to the symptoms produced by this species on radish seedlings including reduction of shoots and root length, radial swelling and tissue chlorosis. The significance of this remains unresolved but it is clear that there is a pathogenicity island containing several genes associated with pathogenicity Bukhaliedet al., (2002). The most known of the genes involved is the nec1 gene which is involved in the thaxtomine A production. This phytotoxin plays an important role in virulence but is not essential for pathogenicity since some strains which do not have the nec1 gene are pathogenic.

The advent of DNA based molecular methods helps to study inter- and intra –specific variation between isolates. In the present study, the RAPD –PCR method was used to study the genetic diversity among Streptomyces isolates. DNA pattern from morphologically similar and dissimilar taxa from the

same and different resources were analyzed in order to establish whether species nomenclature based on host association has any phylogenetic significance and provide new insights to the concept of host- based nomenclature in cultures Jeewon et. al., (2004). RAPD markers have different levels of success to detect molecular marker specific to Streptomyces. Majer, et al. (1996) suggested that RAPD markers lack reproducibility. Although the range of geographic isolates represented in this collection was limited, each isolate examined in this study possessed a unique set of phenotypic characteristics. (McVey and Leonard 1990). Quantifying target microbial populations in complex communities’ remains a barrier to studying species interactions in soil environments Daniel et. al., (2010). There was little correspondence between the phenotypic trails : isolates that were similar genetically were not more likely to be similar in nutrient utilization or inhibition resistance profiles than isolates that were genetically distinct Bramwell et al., (1998) suggested that lack of correlation between inhibition and resistance phenotypes, indicates that isolates of similar inhibitory phenotypes do not have similar resistance phenotypes, indicating that selection for specific inhibition and resistance abilities may be independent . The current study proves that there are different cultural and different isolates in this collection, and indicate that RAPD is an efficient method for discriminating and studying genetic diversity of Streptomyces isolates. Nevertheless, Wanner (2007), suggests that more than 80% of Streptomyces attacks were really attributed to S. scabies. The use of both the phenotypic patterns and the molecular markers revealed excellent tools for the Streptomyces species determination Bouchek- Mechiche et al, 2000; Wanner, (2007).The considerations discussed in the current study affirm that S. scabies is effectively present in all studied potato production regions. Hence, much attention should be taken to understand the biology of the causal agent, and controlling means to decrease the negative consequences of this disease.

Reference

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لعدد من سالالت ستربتوميسس سكابس مسببDNAلتعدد الشكلي في الـ اRAPD-PCRالجرب العادي في البطاطس باستخدام تكنيك



عبده مهدي محمد مهدي*علي السيد توفيق** فتحي جاد محمد* رؤوف نجيب فوزي* أمنيةأحمد عبد الحافظ**

قسم النبات الزراعي- فرع أمراض النبات - كلية الزراعة بمشتهر- جامعة بنها*معهد أمراض النبات – مركز البحوث الزراعية بالجيزة- مصر**

بغ��رض تعري��ف وتوص��يف ع��دد من س��الالت االستربتوميس��س الممرض��ة في مص��ر، تم ع��زل س��بعة وخمسون عزلة من درنات بطاطس وتربة لحقول مصابة بالمرض حيث جمعت من عدة مناطق داخ��ل مص��ر. تم تقسيم العزالت حسب لون المستعمرات والجراثيم وإنتاج الصبغة ومورفولوجب��ا الج��راثيم . وق��د أظه��رت النت��ائج

Modifiedأنه��ا تنتج ص��بغة الميالنين عن��د تنميته��ا على بيئ��ات salt starch agar (MSSA)و.Yeast malt extract (YME)وOat meal agar tyrosine (OMA-T)وقد أوضحت النتائج أن اثنين وعشرون عزلة كانت .

سالبة لصبغة جرام كما أن جميعها قد أحدث إصابة على الجذور والمجم��وع الخض��ري لب��ادرات الفج��ل المخت��برة. كم��ا وج��د اختالف في ش��دة المرض��ية بين الع��زالت عن��د اس��تخدام اختب��ار ال��درنات الدقيق��ة في اختب��ار الق��درة

باس��تخدامDNAاالمراضية. وقد تم تقسيم العزالت حس��ب الش��كل الظ��اهري والنش��وء الن��وعي المب��ني على ال� وذلك للتعرف على االختالف الوراثي لثالثة عش�رة عزل��ة من الميك�روب ستربتوميس�سRAPD-PCRطريقة ال�

والذي يتك��ون p والتي تتكون من عشرة نيكلوتيدات والبادئ6و1سكابيس باستخدام ثالثة من البادئات هي رقم DNA هو األفضل في اظهار التع��دد الش��كلي في ال��1من خمسة عشرة نيوكلتيدة حيث تبين أن استخدام البادئ

فيما بين عزالت ستربتوميسس المختبرة حيث أوضح استخدام هذا البادئ الدرجات العالية في التق��ارب والتباع��دبين جميع العزالت المختبرة.


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