Seyyed Mohsen Taghavi

Citrus canker, caused by Xanthomonas citri pv. citri, is one of the most devastating bacterial diseases of citrus species. The global population of X. citri pv. citri includes three lineages designated as pathotypes A, A*, and A ^w . Whereas pathotype A is the most prevalent lineage around the world, the citrus canker pathogen in Iran includes only pathotype A* strains. Previous work on the X. citri pv. citri strains collected before 2013 showed that two lineages of the pathogen were present in Iran (i.e., 4.1 and 4.4, with 4.4 not present anywhere else at that time). In this study, using a new set of strains collected in 2021 and 2022, we reassessed the population structure of the pathogen in Iran using a phylogeographic approach. All strains isolated in Iran still belonged to pathotype A*. Multilocus variable number tandem repeat analysis revealed that 62 Iranian strains collected between 1991 and 2022 were distributed among 22 haplotypes. Four new haplotypes were identified within the strains isolated in this study that have not previously been reported elsewhere in the world. Although all pre-2013 strains isolated in the Sistan-Baluchestan province of Iran were grouped in subcluster 4.4, and all post-2020 strains isolated in the same area were identified as members of subcluster 4.1. None of the post-2020 strains isolated in Iran belonged to subcluster 4.4, which suggests a shift in the population structure of the pathogen over the past two decades. Our data will pave the way for research on the population structure of the citrus canker pathogen in the area.

Stewart’s wilt of maize, caused by the gram-negative bacterium Pantoea stewartii subsp. stewartii, is one of the economically important diseases of the crop around the globe. The disease has not yet been reported in Iran. Following preliminary observation of suspected symptoms on maize plants in Southeastern Iran in 2019, a 4-year comprehensive field survey and sampling program was initiated across maize-growing areas in the country to monitor the occurrence and distribution of the disease. During 2019 to 2022, 180 maize fields were surveyed, from which 400 symptomatic and asymptomatic maize samples were collected. Among dozens of bacterial strains isolated from leaves and stems of maize plants, 22 strains phenotypically resembled those of Pantoea spp. Species-specific PCR and multilocus phylogenetic analyses using the sequences of atpD, gyrB, infB, and rpoB genes showed that P. stewartii subsp. stewartii is the causal agent of Stewart’s wilt in Iran. All strains induced leaf chlorosis and plant stunting on maize plants under greenhouse conditions 7 to 15 days postinoculation, confirmed with accomplishment of Koch’s postulates. These 22 strains were isolated in Bushehr, Fars, Hormozgan, Kermanshah, Khuzestan, Kohgiluyeh-Boyer Ahmad, and Sistan-Baluchistan provinces. Results of the present study confirmed the occurrence of P. stewartii subsp. stewartii causing Stewart’s wilt of maize in Iran. The occurrence of Stewart’s wilt on maize in Iran can lead to yield reduction, trade restrictions, increased control costs, and threats to food security. Considering the quarantine status of the pathogen in Iran, strict inspections are warranted to combat potential threats due to Stewart’s wilt epidemics in the country.

Microbial plant pathogens possess higher diversity in the center of domestication of their host plants than in the introduced geographic area of the corresponding crops. In this study, the center of wheat, barley, and oat domestication in the Iranian Plateau was surveyed to shed light on the population structure, taxonomic diversity, and biology of Clavibacter species associated with these crops. Comprehensive field surveys completed with pathological assays and molecular-phylogenetic analyses showed that phylogenetically diverse groups of Clavibacter strains were associated with bacterial mosaic symptoms on small grain cereals in this domestication center. Besides the two previously described species, Clavibacter tessellarius and Clavibacter zhangzhiyongii , three clades of atypical Clavibacter strains, phylogenetically distinct from all described Clavibacter species, were isolated from wheat, barley, and oat in this study. Thus, Clavibacter strains associated with small grain cereals in the center of domestication of these crops belong to at least five taxonomically distinct clades, whereas simultaneous occurrence of multitaxa Clavibacter strains on small grain cereals has never been reported outside the Iranian Plateau. All five phylogenetic Clavibacter clades were capable of inducing mosaic and chlorosis on their host of isolation. Whole genome sequence-based comparative investigations showed that most virulence-associated genomic contents of the five Clavibacter clades isolated in this study were somewhat similar. Data obtained in this study suggest a correlation between the center of wheat, barley, and oat domestication and the high taxonomic diversity of the accompanying Clavibacter pathogens. Additional phylogeographic investigations in the area would strengthen this co-evolutionary hypothesis. IMPORTANCE: Members of the gram-positive corynebacterial genus Clavibacter sp. (family Microbacteriaceae ) are seed-borne plant pathogens causing economically important plant diseases on annual crops and vegetables. While comprehensive global-scale studies have been conducted to study the population structure of Clavibacter species infecting tomato, potato, and pepper, phylogenomics investigations have not yet been conducted to monitor the taxonomic diversity, pathogenicity, and origin of Clavibacter strains pathogenic to small grain cereals. Archaeobotanical investigations suggested that human agriculture was established ≈11,000 years ago in the foothills of the Zagros Mountains in Northwestern Iran, while wheat and barley were among the very first crops domesticated in these areas. Thus, the analyses of taxonomic diversity and population structure of Clavibacter strains in the Iranian Plateau would shed light on the correlation between the center of domestication of these crops and the center of diversity of accompanying bacterial pathogens. Our findings showed that most of the Clavibacter strains isolated from small grain cereals in Iran belonged to the two previously described species Clavibacter tessellarius and Clavibacter zhangzhiyongii , while several strains were grouped in three distinct clades, all representing hypothetical novel species in the genus.

Bacterial canker of tomato caused by the gram-positive corynebacterial species Clavibacter michiganensis is one of the most destructive seedborne diseases in both open-air and greenhouse tomatoes. The pathogen is a regulated agent in all tomato-producing countries, as translocation of infected tomato materials transports the bacterium into new areas. C. michiganensis is generally known to have yellow-pigmented colonies on culture media, which is a key differentiative phenotypic feature in standard diagnostic guidelines. During 2020 and 2021, pink-pigmented corynebacterial strains were isolated from tomato seeds (cultivar Sun 6189F1) and plants showing severe canker symptoms in Southern Iran. The six pink-pigmented strains were pathogenic on tomato and pepper seedlings under greenhouse conditions and yielded positive results with C. michiganensis-specific primers pairs described in the literature. Phylogenomics and DNA similarity calculations showed that the pink-pigmented strains were highly similar to the authentic yellow-pigmented members of the pathogen. Thus, they were identified as a new phenotypic variant of tomato bacterial canker pathogen. Whole-genome screenings accomplished with PCR-based assays showed that the pink strains contain all pathogenicity-determinant genes described in C. michiganensis. Further, orthologous gene clusters in the pink-pigmented strains were more similar to the pathogenic members of C. michiganensis than to those of nonpathogenic tomato-associated Clavibacter species. The results obtained in this study demonstrate the emergence of a new pink-pigmented variant of C. michiganensis and highlight the importance of colony pigmentation/morphology in culture-based detection of the bacterium. The need for updating diagnostic guidelines on the colony variants of the pathogen is further discussed.

Taxonomically diverse Pseudomonas species induce bacterial blotch of edible mushrooms around the world. Pseudomonas tolaasii, [P. gingeri], and P. agarici are dominant mycopathogenic pseudomonads in mushroom production farms. In this study, among 216 mycopathogenic bacterial strains isolated from edible mushrooms in Iran, 96 strains were identified as Pseudomonas spp., while only three strains were preliminarily identified as P. agarici. Multilocus sequence analysis showed that only one strain (FH2) authentically belonged to P. agarici, while the other two strains either belonged to [P. gingeri] or represented a unique phylogenetic clade. The three strains also differed from each other in phenotypic characteristics, for example, production of fluorescent pigment and the reaction to tolaasin produced by P. tolaasii. Pathogenicity assays under a controlled environment showed that the symptoms induced by authentic P. agarici were far less severe than those caused by the predominant species P. tolaasii. Furthermore, coinoculation of P. agarici with three bacterial pathogens that are prevalent in Iran on mushroom caps, that is, P. tolaasii, Ewingella americana, and Mycetocola sp., resulted in the development of combined symptoms representing characteristics of both pathogens. The antibiosis assay showed that tolaasin-producing strains of P. tolaasii could inhibit the growth of P. agarici, while tolaasin-negative strains of the same species were unable to do so. This led us to the hypothesis that the inhibitory effect of P. tolaasii on P. agarici is driven by tolaasin production in the former species. This inhibitory effect is also associated with the rarity of P. agarici in natural conditions.

Bacterial blight and leaf spot of geraniums is a destructive disease of cultivated Pelargonium species around the world. During 2020 to 2021, surveys were conducted in seven geranium-growing provinces of Iran to monitor the status of bacterial blight and leaf spot disease. The disease was observed in six surveyed provinces varying in the extent of occurrence and severity. Twenty-two gram-negative pale-yellow bacterial strains resembling members of Xanthomonas were isolated from symptomatic leaves and stems. Pathogenicity and host range assays showed that the bacterial strains were pathogenic on Pelargonium grandiflorum, P. graveolens, P. peltatum, and P. zonale. All strains were positive for a PCR test using the primer pair XcpM1/XcpM2, which is specific for X. hortorum pv. pelargonii. Phylogenetic analysis using the sequences of gyrB and lepA genes showed that the 22 strains clustered in a clade among the sequences of X. hortorum pv. pelargonii strains retrieved from GenBank but were distinct from the other pathovars of X. hortorum. BOX-PCR-based fingerprinting using BOX-A1R primer revealed that the strains isolated in this study were grouped into two clusters, while no distinct correlation was observed between the host/area of isolation and BOX-PCR fingerprinting. None of the strains obtained in this study nor the reference strain of the pathogen produced bacteriocin against each other. Results obtained in this study shed light on the geographic distribution, taxonomic status, and host range of the bacterial blight and leaf spot pathogen of geraniums in Iran, paving the path for further research on disease management.

Key message Whole-genome QTL mining and meta-analysis in tomato for resistance to bacterial and fungal diseases identified 73 meta-QTL regions with significantly refined/reduced confidence intervals. Abstract Tomato production is affected by a range of biotic stressors, causing yield losses and quality reductions. While sources of genetic resistance to many tomato diseases have been identified and characterized, stability of the resistance genes or quantitative trait loci (QTLs) across the resources has not been determined. Here, we examined 491 QTLs previously reported for resistance to tomato diseases in 40 independent studies and 54 unique mapping populations. We identified 29 meta-QTLs (MQTLs) for resistance to bacterial pathogens and 44 MQTLs for resistance to fungal pathogens, and were able to reduce the average confidence interval (CI) of the QTLs by 4.1-fold and 6.7-fold, respectively, compared to the average CI of the original QTLs. The corresponding physical length of the CIs of MQTLs ranged from 56 kb to 6.37 Mb, with a median of 921 kb, of which 27% had a CI lower than 500 kb and 53% had a CI lower than 1 Mb. Comparison of defense responses between tomato and Arabidopsis highlighted 73 orthologous genes in the MQTL regions, which were putatively determined to be involved in defense against bacterial and fungal diseases. Intriguingly, multiple genes were identified in some MQTL regions that are implicated in plant defense responses, including PR-P2, NDR1, PDF1.2, Pip1, SNI1, PTI5, NSL1, DND1, CAD1, SlACO, DAD1, SlPAL, Ph-3, EDS5/SID1, CHI-B/PR-3, Ph-5, ETR1, WRKY29, and WRKY25. Further, we identified a number of candidate resistance genes in the MQTL regions that can be useful for both marker/gene-assisted breeding as well as cloning and genetic transformation.

In 2021, two gram-negative bacterial strains were isolated from garlic (Allium sativum) bulbs showing decay and soft rot symptoms in Central Iran. The bacterial strains were aggressively pathogenic on cactus, garlic, gladiolus, onion, potato, and saffron plants and induced soft rot symptoms on carrot, cucumber, potato, and radish discs. Furthermore, they were pathogenic on sporophores of cultivated and wild mushrooms. Phylogenetic analyses revealed that the bacterial strains belong to Burkholderia gladioli. Garlic bulb rot caused by B. gladioli has rarely been reported in the literature. Historically, B. gladioli strains had been assigned to four pathovars, namely, B. gladioli pv. alliicola, B. gladioli pv. gladioli, B. gladioli pv. agaricicola, and B. gladioli pv. cocovenenans, infecting onion, Gladiolus sp., and mushrooms and poisoning foods, respectively. Multilocus (i.e., 16S rRNA, atpD, gyrB, and lepA genes) sequence-based phylogenetic investigations including reference strains of B. gladioli pathovars showed that the two garlic strains belong to phylogenomic clade 2 of the species, which includes the pathotype strain of B. gladioli pv. alliicola. Although the garlic strains were phylogenetically closely related to the B. gladioli pv. alliicola reference strains, they possessed pathogenicity characteristics that overlapped with three of the four historical pathovars, including the ability to rot onion (pv. alliicola), gladiolus (pv. gladioli), and mushrooms (pv. agaricicola). Furthermore, the pathotype of each pathovar could infect the hosts of other pathovars, undermining the utility of the pathovar concept in this species. Overall, using phenotypic pathovar-oriented assays to classify B. gladioli strains should be replaced by phylogenetic or phylogenomic analysis.

Bacterial leaf streak of small-grain cereals is an economically important disease of wheat and barley crops. The disease occurs in many countries across the globe, with particular importance in regions characterized by high precipitation or areas in which sprinkler irrigation is used. Three genetically distinct lineages of the Gram-negative bacterium Xanthomonas translucens ( X. translucens pv. undulosa, X. translucens pv. translucens, and X. translucens pv. cerealis) are responsible for most of the bacterial leaf streak infections on wheat and barley crops. Considering the seedborne nature of the pathogens, they are included in the A2 (high-risk) list of quarantine organisms for some European countries; hence, they are under strict quarantine control and zero tolerance. Due to the taxonomic complexities within X. translucens, the exact geographic distribution of each pathovar has not yet been determined. In this mini review, we provide an updated overview of the detection and diagnosis of the bacterial leaf streak pathogens. First, a short history of the leaf streak pathogens is provided, followed by the symptomology and host range of the causal agents. Then, the utility of conventional methods and high-throughput molecular approaches in the precise detection and identification of the pathogens is explained. Finally, we highlight the role of quarantine inspections and early detection of the pathogen in combating the risk of bacterial leaf streak in the 21st century's small-grains cereals’ industry.

Bacterial leaf streak disease caused by Xanthomonas translucens pv. undulosa is an economically important disease threatening wheat and barley crops around the globe. Thus far, specific PCR-based detection and identification tests for X. translucens pathovars are not available. In this study, we used comparative genomics approach to design a pathovar-specific primer pair for detection of X. translucens pv. undulosa in naturally infected seeds and its differentiation from other pathovars of the species. For this aim, complete genome sequences of strains of different X. translucens pathovars were compared and the specific PCR primer pair XtuF/XtuR was designed. These primers were strictly specific to X. translucens pv. undulosa because the expected 229-bp DNA fragment was not amplified in the closely related pathovars or in other xanthomonads, wheat-pathogenic bacteria, and other plant-pathogenic bacteria. High sensitivity of the primer pair XtuF/XtuR allowed detection of pure DNA of the pathogen in a concentration as low as 4.5 pg/μl. The pathogen was also detected in water suspension at a concentration of 8.6 × 10² CFU/ml. The PCR test was capable of detecting the pathogen in extracts of naturally infected wheat seeds at a concentration of 3.5 × 10⁴ CFU/g while a culture-plate method was able to detect the pathogen at a concentration of 50 × 10⁵ CFU/g of the same seeds. The PCR test developed in this study is a step forward for precise detection and identification of X. translucens pv. undulosa to prevent outbreaks of the bacterial leaf streak disease.