Multiple Introductions of Tomato Pathogen Clavibacter michiganensis subsp. michiganensis into Iran as Revealed by a Global-Scale Phylogeographic Analysis

Abstract

Tomato bacterial canker caused by Clavibacter michiganensis subsp. michiganensis is one of the most important seed-borne tomato diseases around the globe. The disease was initially reported in 1993 in Iran, and it became a rising threat for the multibillion dollar tomato industry of the country during the last decade. In this study, using phylogeographic analyses, we determined genetic diversity and geographic distribution of C. michiganensis subsp. michiganensis in Iran. Our field surveys showed that the pathogen is expanding into the southern and eastern areas of the country. Furthermore, multilocus sequence analysis and typing (MLSA/MLST) using the sequences of five housekeeping genes (atpD, gyrB, ppk, recA, and rpoB) revealed that 37 C. michiganensis subsp. michiganensis strains isolated in Iran had high genetic diversity and placed in 15 sequence types (STs), while all the available 184 worldwide C. michiganensis subsp. michiganensis sequences were placed in 43 STs. MLSA divided the worldwide C. michiganensis subsp. michiganensis strains into two phylogroups (I and II). Among the 37 strains isolated in Iran, 30 strains clustered in phylogroup I, while 7 strains clustered in phylogroup II. Phylogeographic data inferred from the allelic profile of the five housekeeping genes suggested multiple introductions of C. michiganensis subsp. michiganensis inoculum into Iran, while the geographic origin of the Iranian C. michiganensis subsp. michiganensis strains remains undetermined. Further analyses using higher numbers of strains are warranted to decipher the evolutionary history of C. michiganensis subsp. michiganensis in Iran. Additionally, stricter seed/transplant inspections are recommended to reduce the risk of pathogen expansion to areas with no history of the disease. IMPORTANCE Clavibacter michiganensis subsp. michiganensis, the causal agent of tomato bacterial canker disease, is one of the economically important pathogens of solanaceous crops (e.g., eggplant, pepper, and tomato) around the world. The disease occurs in many countries, with a particular importance in regions characterized by high precipitation and humid environmental conditions. As a seed-borne pathogen, C. michiganensis subsp. michiganensis is included in the A2 (high risk) list of quarantine pathogens by the European and Mediterranean Plant Protection Organization (EPPO). Bacterial canker disease was reported for the first time in 1993 in Iran, while the geographic distribution, genetic diversity, and phylogenetic position of the causal agent remain undetermined. In this study, using the multilocus sequence analysis and typing (MLSA/MLST) approach, we provided a phylogeographic scheme for the C. michiganensis subsp. michiganensis strains isolated in Iran. Furthermore, global-scale phylogenetic analyses led to determination of phylogenetic position of Iranian C. michiganensis subsp. michiganensis strains among worldwide population of the pathogen. Based on diversity parameters and population structure, we suggest relatively higher genetic diversity of the bacterial canker pathogen in Iran than has so far been observed in the other areas of the world. Results obtained in this study provide a novel insight into the genetic diversity and population structure of the bacterial canker pathogen on a global scale.

Comparative Genomics and Phylogenetic Analyses Suggest Several Novel Species within the Genus Clavibacter , Including Nonpathogenic Tomato-Associated Strains

“…Plasmids were isolated according to the procedure described by Kotchoni et al (48) with minor modifications. The tomato-pathogenic C. michiganensis sensu stricto strain ICMP 22049 was used as positive control (49). Bacterial strains were grown in 50 ml Luria-Bertani (LB) medium on a shaker at 150 rpm and 27°C for 48 h. Bacterial cells were harvested by centrifugation at 13,000 ϫ g for 1 min at room temperature, and the pellet was resuspended in 200 l of "solution I" of the protocol of Kotchoni et al (48), mixed well, and incubated at 37°C for 20 min.…”

A centenary for bacterial spot of tomato and pepper

“…Beside the conventional specific/semispecific culture media, serological techniques, DNA fingerprinting methods, and highly specific and sensitive PCR-based techniques are available for the detection of bacterial spot pathogens (Leite et al, 1995;Pečenka et al, 2020). Simultaneous occurrence of bacterial spot and bacterial canker (caused by Clavibacter michiganensis) pathogens in the same tomato field has been recorded (Ansari et al, 2019;Osdaghi, Ansari, et al, 2018). Both of these pathogens as well as a number of tomato-associated nonpathogenic bacteria, for example, epiphytic Curtobacterium flaccumfaciens strains (Osdaghi, Taghavi, Hamzehzarghani, et al, 2018), produce yellowpigmented, domed circular mucoid colonies on culture media, making the initial identification of the causal agent challenging.…”

Screening of eggplant genotypes for resistance to bacterial wilt disease caused by Clavibacter michiganensis subsp. michiganensis

“…Bacterial diseases are one of the major phytopathological problems of solanaceous crops (Davis et al 1984;Sen et al 2015). Cmm is a seed borne pathogen of tomatoes that may cause important crop losses (Yang & Francis, 2007;Ansari et al 2019). Information on the sources of the resistance and its inheritance are necessary to devise efficient and successful breeding strategies for developing resistant cultivars.…”

“…Highly virulent strains of Cmm were isolated from eggplants in Lithuania (Waleron et al 2011) and in Turkey (Kara et al 2018;Sen et al 2018). These virulent strains have a considerable risk to be potentially spread globally (Ansari et al 2019).…”