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RESEARCH JOURNAL OF FISHERIES AND HYDROBIOLOGY
© 2015 AENSI Publisher All rights reserved
ISSN:1816-9112
Open Access Journal
Copyright © 2015 by authors and American-Eurasian Network for Scientific
Information.
This work is licensed under the Creative Commons Attribution International License
(CC BY). http://creativecommons.org/licenses/by/4.0/
Phylogenetic diversity of Iranian strains
of Rhodococcus fasciansby 16srDNA
analysis
Kavian, S. and Najafipour, G.
Plant Pathology Department,
Jahrom Branch, Islamic Azad
University, Jahrom, Iran
Address For Correspondence:
A Najafipour, G., Plant Pathology
Department, Jahrom Branch,
Islamic Azad University, Jahrom,
Iran
E. mail: [email protected]
and [email protected]
Received: Feb 19, 2015
Accepted: May 18, 2015
Published: May 25, 2015
ABSTRACT
Rhodococcusfascians is a gram-positive and phytopathogenic bacterium which can simulate
tumor formation in many monocotyledons and dicotyledons. In the present study, 24 isolates of
Rhodococcusfascians were collected from different regions of Fars province (Shiraz, Jahrom,
Darab, Fasa, Laar and Abadeh). These isolates were examined for phenotypic characteristics.
Also, 4 isolates of geranium were assessed for phylogenicfeatures using 16s rDNA primers.
Examination on phylogenic characteristics via using 2 primers designed from 16s rDNA showed
thatRhodococcusfascians isolates belonging to geranium of Abadeh, Shiraz, Jahrom and Darab
are in a sister group with isolates derived from Gene Bank with more than 99%
homology.Geranium strains of Shiraz and Darab showed the maximum and minimum similarity
with sequences existing in the Gene bank, respectively.Also, the results indicated that isolates of
Jahrom (2), Darab (16) and Abadeh (10) are in the same group, and they are distinguished from
the isolates of Shiraz (4). In this group, there are two subgroups in which the isolates of Jahrom
and Darab were placed in the same subgroup and Abadeh isolate (10) was placed in another
subgroup.It seemssimilar climate (hot and dry) in Jahrom and Daraband geographical proximity
are reseons for their genome homology. The results obtained show that using genotypic features,
different geographic regions can be distinguished from each other.
KEY WORDS: Rhodococcus fascians, Fars province, Leafygall, Ornamental plants.
INTRODUCTION
Rhodococcus fascians is the causual agent of leafy gall, flat limb and malformation in the stems of many
mono- and dicotyledons. In the first time, in 1927, disease was reported in sweet pea, by Neil Brown. Several
plant species in mono-and dicotyledonous plants are at risk of infection with Rhodococcus fascians. The
symptoms which can be naturally found in plants include proliferation of buds in the leaf axis which sometimes
described as brooming, abnormal growth of branches in the crown of plant, producing of adventitious roots, or
in some cases, root growth stopping.
Taxonomic place of Rhodococcus fascians had been changed during years. In 1984, according to the
phenotypic, genotypic and biochemical informations, the bacterium was placed in Actinomycetales and
Rhodococcus genus, and it was named as R. fascians (Goodfellow, 1984). Recently, according to the taxonomic
informations, members of Rhodococcus spp. have been located in corynebacterineae (which produces mycolic
acid), family Nocardiaceae and phylum Actinobacteria. This genus is consisting of gram-positive bacteria with
high G+C content. Also, it has been recommended that the classification of Nocardiaceae is modified at the
base of 16srDNA sequence (Zhi et al, 2009). 16SrDNA sequencing is one of the best methods to phylogenic
study and comparision of closed bacteria. Also it can be used as a reliable method for identification of many
bacterial species via PCR-based techniques or nucleotide markers.
In Iran, regarding to disease report in Fars province, there is a few comprehensive studies related to the
genotypic status of R. fascians. So, the purpose of the present study was to examine the genetic diversity of
representative isolates in Fars province using determination of 16SrDNA sequenceing.
METHODS AND MATERIALS
Examination on phenotypic characteristics of isolates:
During 2013 to 2014, several regions in Fars province (Shiraz, Darab, Jahrom and Abade) were
investigated and ornamental plants showing leafy gall were collected and transferred to the laboratory. First, the
RESEARCH JOURNAL OF FISHERIES AND HYDROBIOLOGY, 10(9) May 2015, Pages: 253-257
Kavian, S. and Najafipour, G., 2015
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samples were washed with tap water, renised twice with sterile distilled water and ground in a small
amount of sterile distilled water. A loopful of suspension was streaked on nutrient agar (NA) and
incubated on 27ºC. Three to five days after culturing, small orange or yellow colonies, slightly or non-mucoid
were selected and purified. 24 isolates were purified and physiological and biochemical tests were performed on
them, according to standard bacteriologicalmethods (Fahy and Parsley, 1983; Schaad et al., 2001).
DNA Extraction:
Sample Preparation:
Bacterial isolates were grown on NA medium at 27oC for three days. A loopful of each isolate was
suspended in steril distilled water to a concentration of 107CFU (OD 600 =1) (Klambt et al., 1966). Sterile
distilled water was used as negative control. Alkaline lysis method was used to DNA extraction.10 µlof each
bacterial suspension were mixed with 100 µl of 0.05-mol NaOH and it was placed at 95oC for 15 minutes. The
suspension was centrifuged at 14000 rpm for 2 minutes; the supernatant was transferred to clean tubes.
Genomic samples of DNA were stored at -20oC (Rademaker et al. 1997).
PCR and Nucleotide sequencing of isolates:
In order to determine the phylogenic position of bacteria using 16srDNA, p0 (5’GAGAGTTTGATCCTGGCTCAG-3’) and p6 (5’-CTACGG CTACCTTGTTACGA-3’) primerswere used
(Picard et al., 2000).These primers synthesizes an approximately 1500bp DNA fragment in size.The PCR
reaction was performed in Bio-Rad I-cycler (USA) in 20 µl PCR mixture, consist of 2 µlof DNA
template, 2 µl of 10X Buffer PCR, 1 µl of dNTP, 1 µl of each primers, 0.5 unit of Taq DNA Polymerase. The
PCR reaction was carried out for 1 cycle as a primary denaturation 95°C for 5 min, followed by 35 cyclesas the
following condition: 95°C for 30sec, 50°C for 30 sec and 72°C for 4 min and final extension was 72°C
for 10 min (Picard et al., 2000). The PCR products were electrophoresed on 1.5 % TBE agarose gel at 75V/cm
for 1.5 hours. The gels were stained with ethidium bromide, viewed and photographed under
UVillumination (Bio Rad, USA).
Fig. 1: Electrophoretic patterns of PCR products using P0 and P6primersof 16srDNA.
M: DNA Marker;1-4 geranium isolates:lane1, negative control; lane2,Jahromisolate (2);lane3, Shiraz
Isolate (4);lane4,Abadeh isolate (10);lane5,Darab isolate (16).
Computer Analysis of Sequences:
PCR products of four geranium isolates, collected from different regions of Fars province, were sequenced
(Macrogen Co., Korea). Sequencing results were modified using Seqman software and aligned using Blast
program with four sequences in Gene bank. Moreover, one sequence of Clavibacter michiganensis used as
outgroup.Target sequences were aligned with data bases in NCBI using ClustalW Algorithm. Phylogenic tree of
isolates were drawn using Mega-5 software and Neighbor-Joining method.
Results:
All isolates were positive in gram reaction and catalase, and negative in oxidase reaction, arginine
dehydrolase, levan formation, starch hydrolysis and hypersensitive test on geranium. In addition, isolates were
obligate aerobes and had the ability to produce H2S from peptone. On the basis of biochemical and phenotypic
tests, all isolates recognized as R. fascians (data not shown).
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The results of 16srDNAsequencing showed that geranium isolates of Abadeh (10), Shiraz (4), Jahrom (2)
and Darab (16) are homogenous and all fell to one cluster. Moreover, four sequences of Rhodococcus fascians
obtained from Gene bank, showed only 0.2% variance with Fars’s geranium isolates; therefore they are placed
in the same cluster too (fig 2).
Fig. 2: phylogenic dendrogram derived from the comparison of gene sequence of 16srDNA belonging to 4
strains of Rhodococcusfascians isolated from geranium in Fars with a number of Rhodococcus fascians
and R. rhodochorus isolates obtained from gene bank. Clavibacter michiganesis has been used as out
group too.
Schloss and collagues (2005) proposed that in phylogenic analysis, bacteria which having more than
97%similarity in 16srDNAsequencing, must considered as the same species.
Outgroups (R. rhodochrous and Clavibacter michiganensis) had 93% and 94% similarity to R. fascians,
respectively. Besides, the isolates of 10, 2, 4 and 16 had homology of 94.4% with C. michiganensis.These
results were not unexpected because different species must have less than 97% similarity to each other (Brunt
and Goobell, 1994).
Discussion:
Our findings showed that leafy gall disease is scattered in all sampeling regions in Fars province (Iran).
Symptoms include the proliferation of buds in leaf axis, abnormal producing of branches at the base of stems,
thick leaves and branches, gall-like masses at the base of plant or cutting collected, that are very similar to the
symptoms reported by Lacey and coworkers (1936).
In the present study, most isolates of geranium (4 isolates of 7 isolates) were placed in cluster 1. Also,
petunia isolates were often in cluster 2. So, it seems that nevertheless the distribution of petunia and geranium
isolates in different group, the most of them were arranged in clusters 1 and 2, respectively.
By this method, isolates belonging to different hosts may be distinguished from each other. In another
study, Najafipour and Taghavi (2001) examined the phenotypic, biochemical, pathogenic characteristics and
host range of different strains of Rhodococcus fascians isolated from different regions in Shiraz, and compared
them with the strains of Northern of Iran. Their results demonstrated that the isolates from various hosts in
Shiraz have very high homogeneity with each other. Their results differ from the results obtained in present
research. It may be due to the limitation of their sampling to Shiraz.
Despite the various biochemical and phenotypic tests to differentiate various isolates from each other, the
existence of some strange characteristics in some cases has made impossible bacterial identification. Genome
sequencing has more advantages in comparison with examination of phenotypic data including easy, more
reliability and more accurate of the results. One of the genome fragments which used for this purpose is
16srDNA. It has been known as one of the most useful and functional molecular methods. This part of genome
has a high degree of functional stability, and it exists in all organisms.In addition, different positions are
changed in their sequences, and they provide conditions that maximum phylogenic relation (maximum genetic
distances) can be measurable.
In the present study, a part of 16srDNA gene from four geranium isolates (2, 4, 10 and 16) was synthetized
using P0 and P6 primersand sequenced. Their sequences were recorded in Gene bank with the accession
numbers: KM262808, KM262807, KM262806 and KM262809, respectively.
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Phylogenic tree of mentioned isolates was drawn using Mega-5 software, neighbor joining method and
1000-replicates Bootstrap.According to the resulteddendrogram, the four our isolates and several sequences of
Rhodococcus fascians, obtained from gene bank, were placed in a sister group with more than 99% homology.
At the base of the suggestion of Stuck Brunt and Goobell (1994), high similarity of 97% shows that isolates
belong to the same species. Therefore, in the recent research, four our isolates belong to Rhodococcus fascians.
In this study, the isolates of R. rhodochrous and C. mishiganensiswere placed in 2 separate groups, and
they have similarity of 93% and 94.4% with R. fascians, respectively.
Although Fars geranium isolates and sequence of Rhodococcus fascians obtained from gene bank were
placed in one sister group, these isolates have not the similarity of 100%. For example, the isolates of 4 and 16
showed the maximum and minimum similarity with sequences existing in gene bank, respectively. In addition,
the dendrogram indicated difference and heterogeneity among the strains of geranium in different regions of
Fars.
This result is consistent with the results obtained by Najafipourand coworkers (2014). They analyzed the
phenotypic characteristics of isolates of geranium, petunia, nasturtium and chrysanthemum in Shiraz,
usingNtsys-pc (v2.2), and found that geranium isolates has many diversity in terms of phenotypic
characteristics.
According to the genotypic dendrogram, isolates of Jahrom (2), Darab (16) and Abadeh (10) were in the
same group, and they were distinguished from the isolates of Shiraz (4). In this group, there are two subgroups
in which the isolates of Jahrom and Darab were placed in the same subgroup and Abadeh isolates (10) were
placed in another subgroup.It is likely that similar compatibility is created in their genome because of similar
climate (hot and dry) in Jahrom and Darab and geographical proximity to each other.
In another research, Sandberg (2007) examined the sequence of 16srDNA belonging to 84 isolates of
Rhodococcusfascians, and showed that there are no significant relation between phylogeny and host type. Since
in our research, single host examined in the study of genotype was geranium and other hosts were not being
examined, therefore, theses results can not be compared with each other.
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