Ralstonia solenacearum is a soil bacterium with devastating effects on many solanaceous crops such as tomato, in which produces the bacterial wilt (also known as brown rot in potato and granville wilt in tobacco). Resistant varieties are currently only available for breeders in tomato, pepper and eggplant, which preoccupies farmers all over the world. Using techniques to monitor the spread of infection in live plants, researchers from the Centre for Research in Agricultural Genomics (Barcelona, Spain) and the North Carolina State University (NC, USA) have recently discovered that tomato resistant varieties are able to stop the bacteria expansion in four different spatiotemporal phases: at the root, in the vertical transport from roots to shoots, in the diffusion among the vascular bundle and, surprisingly, in the colonization of the tissue surrounding the vessels.

 "Ralstonia solanacearum and other vascular pathogens pose a major problem because efforts to date to discover the genes that determine plant resistance to these pathogens have not yielded transferable results in the field, and indicate that resistance is a complex character determined by the interaction of different genes. It was therefore necessary to take a step back and understand how the bacterium behaves in resistant and susceptible plants", explains Núria Sánchez Coll, head of the CRAG research group Bacterial plant diseases and plant cell death and one of the senior authors of the article published in the Journal of Experimental Botany.

“It can be compared to the research needed to treat a new human disease, for example, in Covid-19 we are seeing that the virus infects the lung, but also the kidney, and other cells, and that there are people that are more susceptible than others ... With Ralstonia we also had to understand how the pathogen behaves inside the plant, how it is transported from one organ to another, etc.”, adds the University of Barcelona researcher at CRAG Marc Valls, who co-directs the research group and the study with Sánchez Coll.

This work sets the foundations to decipher the molecular mechanisms that limit pathogen colonization by the plant, which may provide new precision tools to fight bacterial wilt in the field.

Ralstonia solanacearum: a quarantine bacterium

Overall, R. solanacearum is considered one of the most important plant pathogens due to the large number of species it affects, its broad geographical distribution, and its persistence in soil and water. The disease it causes is endemic in tropical and subtropical areas. One particular strain has been classified as Race 3 biovar 2 (R3bv2) and is particularly aggressive on many host plants and can be successful in cooler climates. In the European Union, where some outbreaks have been reported, R3bv2 has been classified as a quarantine pest, and therefore it is subject to strict surveillance.

R. solanacearum infection produces bacterial wilt. The pathogen, present in the soil, enters the plant through the roots and colonizes the vessels that carry water and mineral salts to the aerial parts of the plant, making a plug that causes a rapid wilting of the leaves and, finally, the death of the plant. This mechanism is shared with other vascular pathogens that also invade the xylem of the plant, such as the bacterium Xylella fastidiosa, which currently threatens the crops of olive, almond, grapes, and other species in the Mediterranean.

Few resistant solanaceous options

The vast majority of commercial tomato plants are susceptible to Ralstonia solanacearum and the few resistant varieties that exist make very small tomatoes. In regions where R. solanacearum is a problem, it is common to use commercial varieties which are grafted on rootstocks of the resistant variety. This is precisely the solution implemented by farmers in the state of North Carolina (USA), where bacterial wilt has been reducing profits for multiple crops for more than a century.

“Considering the magnitude of the problem, we started addressing the issue by breeding approaches about a decade ago. While the level of resistance in tomato breeding lines has been improved significantly, combining tomato fruit quality along with bacterial wilt resistance is still a challenge”, says Dilip Panthee, Associate Professor and Tomato Breeder at North Carolina State University, who also contributed to the study.

In an attempt to better understand the characteristics of the plant that determine its resistance to R. solanacearum, researchers at NC State University contacted the CRAG research group led by Valls and Sanchez Coll. The collaboration between the former, accustomed to applied field research, and the latter, experts in fundamental laboratory research, facilitated the discovery of the bottlenecks that resistant tomatoes impose on the bacterium in order to prevent colonization.

Four bottlenecks for the bacterium

To track the process of R. solanacearum infection in tomato plants in vivo, CRAG and NC State University researchers used modified luminescent and fluorescent bacteria. With these bacteria they infected different commercial tomato varieties: some very susceptible (Marmande variety), some of moderate resistance (Shield), and the very resistant plants that are used to make the grafts (Hawaii 7996).

Following the colonization of R. solanacearum in these three varieties, they found that the bacterium's ability to invade plant tissues was different.

“Resistant plants have the ability to block the spread of the bacterium at four different points: in the root, where the bacterium fails to expand, in the vessels that carry water and mineral salts, in the horizontal movement in the bundle of vessels of the vascular system and, finally, in the radial movement of the bacterium from the vessels to the adjacent tissues,” explains Marc Planas-Marquès, CRAG doctoral student and co-first author of the article.

This is the first time that the infection process has been studied so systematically, and researchers have been surprised to find that Ralstonia solanacearum also has the ability to come out of vessels to infect nearby tissue and host resistance can block this invasion.

“It is a very relevant discovery, now we know that, when the infection is very advanced the bacterium leaves the vessel to feed on cortex cells, which can give more capacity for multiplication and spread to other plants”, explains Marc Valls.

“Ultimately, host resistance needs to be combined with field management plans to reduce losses. This work helps us better understand how the pathogen causes damage in the host and should offer clues of how to breed tomatoes with superior host resistance,” observes Frank Louws, Professor at North Carolina State University.

“This work has exciting implications for resistance breeding because now we have a unified context that describes the plant-pathogen battlefields. Also, this research developed new tools and techniques to measure the back-and-forth dynamic at each zone of the bacterial wilt tug-of-war. Resistance mechanisms can be measured more directly throughout the entire plant, which will allow future studies to link each mechanism with the causal genetics,” adds Jonathan Kressin, co-first author of the study.

Returning to the simile of human diseases, researchers now know which organs the pathogen affects and which characteristics define the most resistant hosts. In this sense, it is important to note that the work has been done in commercial varieties. Based on these results, it will be necessary to return to molecular and genetic work to discover the genes that control aspects of resistance and remain very vigilant to contain the spread of this pathogen in our fields.

 

Article of reference: Marc Planas-Marquès, Jonathan P Kressin, Anurag Kashyap, Dilip R Panthee, Frank J Louws, Nuria S Coll, Marc Valls. Four bottlenecks restrict colonization and invasion by the pathogen Ralstonia solanacearum in resistant tomato. Journal of Experimental Botany, 71(6), 2157–2171. DOI:10.1093/jxb/erz562

About the authors and the funding of the study: This work is fruit of a collaboration among researchers from the Centre for Research in Agricultural Genomics (CRAG, Barcelona, Spain) and from the North Carolina State University (USA). The work at CRAG has been funded by grants from the Spanish Ministry of Economy and Competitiveness and the Government of Catalonia and by International Fellowship-Indian Council of Agricultural Research. The authors from at NC State University have been funded by the US National Institute of Food and Agriculture award #2016-51181-25404, the North Carolina Tomato Growers Association and by Monsanto Graduate Fellowship.