Resistance to Candidatus Liberibacter solanacearum in tomato landraces from Mexico

Candidatus Liberibacter solanacearum (CLso) is an economically important plant-pathogen of tomato (Solanum lycopersicum) crops in the United States, Mexico, Central America, and New Zealand. Currently, there are no reports of resistance to CLso in tomato cultivars. Identification and development of CLso-resistant cultivars may offer the most efficient way to manage this tomato disease. Resistance of 46 tomato landraces collected in different regions of Mexico, representing a wide range of genetic variability from this country was evaluated. Two assays were done in consecutively years to assess the resistance to CLso under greenhouse conditions. Plants from both tests were inoculated with CLso through 20 Bactericera cockerelli insects per plant. In the first trial, landraces FC22 and FC44 showed a significantly higher proportion of resistant plants, less symptoms severity, and longer incubation time, followed by landraces FC40 and FC33 compared with the rest of the 42 landraces and 2 susceptible cultivars 60 days post inoculation (dpi). In the second assay, only landraces FC22 and FC44 had again significantly higher proportion of resistant plants, less symptoms severity, relative lower CLso titers, and longer incubation time in comparison with landraces FC40 and FC33 and the two susceptible cultivars 60 dpi, corroborating their resistance to CLso. Presence of CLso DNA in all resistant plants from both assays discards scape plants and indicates that the methodology used was adequate to discriminate between resistant and susceptible plants. These results confirm that landraces FC22 and FC44 are promising resistant sources for the development of CLso-resistant cultivars of tomato. Author summary The bacterium “Candidatus Liberibacter solanacearum” (CLso) is an important plant-pathogen of tomato crops in the United States, Mexico, Central America, and New Zealand. Tomato growers are lacking of cultivars with resistance to this pathogen and the development of resistant cultivars of this crop would make a sustainable business for these growers and healthy tomato consumption for humans. Tomato landraces from countries that are center of domestication of cultivated crops like Mexico, are potentially sources of resistance to plant-pathogens. Therefore, two tests were done looking for resistance sources to this pathogen and we found two tomato landraces (FC22 and FC44) showing high level of resistance to CLso because they had significantly higher resistant plants, less symptoms severity, lower CLso DNA concentration, and delay of the first symptoms in the inoculated plants in comparison with the two commercial cultivars and 44 tomato landraces collected from Mexico 60 days post infection. These landraces are promising resistant sources for the development of CLso-resistant cultivars of tomato.

carota, Apium graveolens, and Pastinaca sativa [1,2,10,11]. 72 In the last 15 years, CLso, which was first described from leaves of Solanum tuberosum 73 plants in New Zeland [12], has become more important worldwide for its high aggressiveness, 74 increasing geographical distribution, wide host range, and because no commercial cultivars of 75 potato and tomato resistant to this bacterium have been reported so far [1,8,10,11,13]. In North 4 76 and Central America, the main symptoms of this disease in tomato plants are the following: 77 overall chlorosis, severe stunting, leaf epinasty, leaf filimorphism or elongated leaves, leaf 78 rolling, crispy leaf, purple discoloration of veins, excessive branching of axillary shoots, flower 79 abortion, and deformation of the fruits [1][2][3][4]14]. 80 The management of this bacterium has been based mainly on the chemical control 81 through the use of insecticides against the vector insect. This method has been partially 82 effective, costly, and represents a biohazard [15,16]. Furthermore, this method can also 83 contribute to the development of resistance in vector populations [17]. An effective alternative, 84 without bio-risk, and accepted for the integrated management of CLso is the development of 85 genotypes resistant to this group of pathogens [13,16,18,19]. In tomato crops, control of other 86 diseases, like TYLCV, transmitted by vectors, through resistant cultivars has been widely used 87 successfully around the world [21]. 88 The first step for the development of disease-resistant cultivars is the screening of wild 89 and/or domesticated genetic resources, to be used afterwards in the genetic breeding programs 90 of agricultural crops [20]. The second desirable step is to analyze the genetic base and 91 heritability of the target trait to design the best breeding model for the introgression of the 92 desirable trait into cultivated background, and the third step would be to carry out the plan [ However, no tomato cultivars resistant to this bacterium have been described in Oceania, 105 Central and North America so far. This could be due to the lack of new sources of resistance 106 and/or studies on the genetic basis of the resistant trait to CLso. Therefore, it is important to 107 continue looking for new sources of resistance that support breeding programs in the 108 development of tomato resistant cultivars to this important pathogen.

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The objective of the present study was to identify resistance sources against CLso using

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CLso identification 115 The samples taken from the inoculum source of tomato plants were positive for the 116 amplification of the predicted 1168 bp fragment corresponding to the CLso bacterium (Fig 1).

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The sequence of the amplified fragment showed 100% nucleotide identity with the KF776420 118 isolate of GenBank corresponding to a sample of CLso-infected B. cockerelli from Guanajuato

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[39] and 99% nucleotide identity with isolates KF776422, EU918197 and FJ939136 tomato 120 plants infected with this bacterium from the state of Sinaloa, and Texas (USA), respectively.

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The phylogenetic analysis of the CLso isolated sequence from the inoculum source grouped 122 the strain used in this study within the CLso species, and clearly differentiated it from other 123 species of the genus Candidatus Liberibacter, as well as from their respective vectors (Fig 2). varied significantly for all these parameters among genotypes in this assay (P < 0.0001) " Table   128 1". Landraces range of symptoms of 1-3, 1-3, 3-9, and 3-9, respectively, whereas the rest of the genotypes 142 had a range of symptoms of 7 -9 "  Reserva which had the shortest delay in developing the first symptoms of CLso " Table 2".    Authors thank FitoCiencia, for the support provided to this research.