First Report of Arthrobacter agilis Associated with Elm Trees in Iran

Abstract

Elms (Ulmusspp.) are popular species of deciduous trees belonging to the family Ulmaceae. Elms are ornamental trees commonly used in cities and forests. During late June 2015, isolation of bacterial associated was performed for the first time on elm trees, in the Tabriz (East Azarbaijan Province, Iran). Based on morphological, biochemical and molecular characteristics, the isolates were identified as Arthrobacter agilis. This study provides the first report on the occurrence of this bacterium on elm trees in Iran.

Keywords: Ulmaceae; Elm trees; Arthrobacter agilis; Tabriz

Case Presentation

Elms are dicots that taxonomically belonging to genus Ulmus, family Ulmaceae, order Rosales and kingdom Plantae. These first appeared in the Miocene geological period about 20 million years ago, originating in what is now central Asia and spread all over the Northern hemisphere over time [1]. There are not precise information about all elms in whole of the world, but estimated that there are 136 million elms whit more than 10 cm diameter that 95 percent of those are belong to Europe, Asia and North America [2]. Iran, also, is one of the countries that some species of elms outspread in it, but the precise map of number and dissemination is not available yet [3]. Some special abilities, including tolerance to salt, wind and place switching, partly fast growing in any kind of soils, wealthy timber, tolerance against physical pressure and soil Compaction, and besides, considerably beauty and elegance because of high height and special form of leaves and branches cause these trees used all over the Europe, North America and other areas of world, day by day [4]. Elms are ornamental trees commonly used in cities and forests of Northwest of Iran.

The tissues of 14 elm trees were collected during 2015-16 from different areas of Tabriz city. To isolate the bacteria from branches and trunks, small sections (10 to 15 mm square) were excised from the margins and centers of these parts. These sections of tissues were sterilized in 70% alcohol for 30 seconds and were rinsed three times in sterilized distilled water for 30 seconds. The tissue pieces were cut, allowing to release bacteria into sterilized distilled water for 1 hour, then 1 ml of the diluted bacterial cell suspension streaked onto Nutrient Agar (NA) medium. The inoculated plates were incubated at 28ºC for 72 hours. Fifty five bacterial strains were obtained and characterized from this work. Observations and continuation the research were made for development of well separated red, coccoid, small bacterial colonies on the nutrient agar medium that they were nine strains.

Hypersensitive reaction and pathogenicity tests were carried out tobacco (Nicotianatabacum) and elm young shoots, respectively. Scratched parts were inoculated with 1 loop full of 48-h old bacterial culture on nutrient agar medium. Parts injected with bacteria strains covered by parafilm. All treatments were conducted with two branches per bacterial isolate. Controls were inoculated with sterile water without bacterial colonies. The inoculated young shoots subsequently incubated at 24ºC for 50 days in the humidity chamber. All of the nine selected strains were used for pathogenicity test.

Biochemical tests, such as gram reaction, catalase, oxidase, urease, lipase, gelativase, aerobic and anaerobic, as described by Schaad et al. (2001) were utilized to characterize the bacterial isolates. The amplification of 16S rDNA was performed with a final volume of 37μl containing 2.5μl of total DNA, 1.5μl of the 27F primer (5′-TCCGTAGGTGAACCTGCGG-3′), 1.5μl of the 1492R primer (5′ TCTCCGCTTATTGATATGC-3′), 12.9μl of distilled water, 18.6μl of master mix [5]. Master mix involve three components that were dNTPs, MgCl₂ and Tag DNA Polymerase. The conditions of PCR were as follows: 95ºC for 5 min, followed by 36 cycles of denaturation at 94ºC for 1min, annealing at 60ºC for 1 min and extension at 72ºC for 1 min, then an extra extension at 72ºC for 10 min. The identification of these isolates was carried out using BLAST (Basic Local Alignment Search Tool) in NCBI, so that the most similarity of experimental sequences verified with the reference sequences in the databases. Since these nine strains were similar in Phenotypic characteristics, one strain (M7) selected for DNA sequencing.

Bacterial colonies, which were isolated from tissues, were red and coccoid-shaped on NA medium. Several isolates of purified colonies were selected for streaking on Nutrient Agar (NA) slants and stored at 4ºC in refrigerator for future use. The phenotypic characteristics of the all isolates were similar to those previously described by Koch et al. [6].

All strains were Gram-positive, oxidase-positive, catalasepositive, urease-negative, gelativaze-positive and lipasse-negative. These bacterial strains weren’t able to produce Hypersensitive Reaction (HR) signs on tobacco leaves. After 50 days of incubation in the humidity chamber, none the young shoots inoculated with bacterial strains have pathogenicity symptoms. Control young shoots remained symptomless, too. Thereby, hypersensitive reaction and pathogenicity test were negative ontobacco and young shoots in all the isolates, respectively (Table 1).

  

    
Characteristic 
    
M7 
    
Arthrobacteragilis 
    
A. flavus 
  
  

    
Colony colour
    
Red
    
Red
    
Yellow
  
  

    
Form
    
Coccoid
    
Coccoid
    
Rod-coccus
  
  

    
Gram reaction
    
Positive
    
Positive
    
Positive
  
  

    
Catalase
    
+
    
+
    
+
  
  

    
Oxidase
    
+
    
+
    
-
  
  

    
Urease
    
-
    
-
    
-
  
  

    
Lipase
    
-
    
-
    
+
  
  

    
Gelativase
    
+
    
+
    
+
  
  

    
Aerobic
    
-
    
-
    
+
  
  

    
Anaerobic
    
-
    
-
    
-
  
  

    
Some    data from Reddy et al. (2000).
      
 
  

Table 1: Phenotypic characteristics of M7 (Arthrobacter agilis) compared with
Arthrobacterflavus +, Positive and - , Negative.