Identification of stable sources of rate-reducing resistance to Septoria lactucae in lettuce germplasm

Septoria leaf spot, caused by Septoria lactucae, is one of the major open-field diseases of lettuce (Lactuca sativa) in tropical and subtropical regions. However, there are few available studies dealing with the identification of useful sources of resistance to this disease. Here, a genetically diverse collection of 42 lettuce accessions was evaluated for Septoria leaf spot reaction under open-field conditions (natural inoculum) during the rainy season. The experimental design was randomized blocks with three replications (16 plants each). Although no immunity-like source was detected, 21 accessions displayed superior levels of rate-reducing resistance with significant lower Area Under Disease Progress Curve (AUDPC) values, encompassing seven accessions of the ‘Crispy green loose-leaf’, eight of the ‘Crispy purple loose-leaf’, two of the ‘Cos/Romaine’, two of the ‘Loose-leaf super crispy’, one accession of the ‘Butterhead’, and one accession of the ‘Iceberg’ morphotype. A subgroup of nine accessions with the highest levels of field resistance was also inoculated under greenhouse conditions with four S. lactucae isolates (collected in distinct geographic areas) in two assays at two distinct phenological stages. The cultivars ‘BRS Mediterrânea’ and ‘Vanda’ displayed lowest AUDPC values at the late vegetative/reproductive phenological phase. The remaining accessions displayed intermediate values of AUDPC, with the exception of the cultivar ‘Rubi’ (control), which exhibited moderate levels of susceptibility. The availability for lettuce breeding programs of genetic sources with higher and stable levels of resistance to S. lactucae will enable the development of more adapted commercial cultivars, increasing the sustainability of this crop across Neotropical regions.

The phenological cycle of lettuce is subdivided into four main stages: germination, transplantation, vegetative, and reproductive phases.During each of these stages, multiple diseases can affect the development of lettuce plants.Septoria leaf spot has been a very common disease in the vegetative phase across lettuce-producing regions with mild temperatures (20-24 °C) and in places with rainy weather or sprinkler-irrigation systems (Blancard et al., 2006;Cabral et al., 2022).The disease is more severe in the transition from the vegetative to the reproductive stage, and can often affect stems and floral organs in seed production fields (Bedlan, 1999).The first symptoms are observed on older leaves, being characterized by small, irregular, chlorotic spots bordered by the leaf veins.These spots, as they evolve, might become necrotic and surrounded by a yellowish halo.Lesions can coalesce and affect the entire leaf area, causing total leaf blade blight and premature leaf fall.Under high moisture conditions, pycnidia are formed and a mass of filiform, multiseptated, and hyaline conidia is observed in the upper portion of these structures.Conidia can spread over short distances via wind and/or water splashes, and host invasion occurs via stomata (Bedlan, 1999).The initial inoculum of S. lactucae is often originated from infested/infected seeds, crop residues or from nearby affected fields.The use of fungicides in conjunction with other disease management measures has not been sufficient to reduce the fungal damage, especially when environmental conditions are conductive to the causal agent (Cabral et al., 2022).
Although Septoria leaf spot of lettuce has been reported to be induced by a complex of fungal species of the genus Septoria in other continents (Bedlan, 1999;Lohmeier et al., 2013), recent molecular characterization of a wide range of isolates indicated Septoria lactucae Passerini (family Mycosphaerellaceae) as the sole causal agent of this disease under Brazilian conditions (Cabral et al., 2022).The identification and incorporation of resistance/tolerance factors to S. lactucae into commercial cultivars is a central strategy for Septoria leaf spot management as well as other seed-borne pathogens (Cabral et al., 2019).In this context, the main objective of the present study was to search for useful breeding sources of S. lactucae resistance in a genetically diverse lettuce germplasm collection.

Location of the field and greenhouse assays
All experimental activities were carried out in the field, greenhouses, and laboratories of Embrapa Vegetables (CNPH), located at an altitude of 996 m above sea level and geographical coordinates of 15°56′00" South latitude and 48°08′00" West longitude, in the Administrative Region of Gama in Brasília-DF, Brazil.

Screening germplasm accessions from distinct lettuce morphotypes to Septoria lactucae with natural inoculum under open-field conditions
The field experiment was carried out from January to March 2014, arranged in randomized blocks, with 42 treatments (lettuce accessions) x three replications (plots).The plots consisted of sixteen plants in a spacing of 0.30 × 0.30 m.The seedlings were produced in styrofoam trays with 128 cells filled with sterilized substrate (Plantmax ® ) and transplanted (after 21 days) into beds fertilized with simple superphosphate.Over time, three topdressing fertilizations with urea (10 g/ m 2 ) were carried out at ten, 20, and 30 days.Infestation with the inoculum of S. lactucae occurred naturally from old lettuce fields located in the vicinity of the experiment.Evaluations were performed by visual analysis of the leaf damage (= severity) induced by the pathogen at 50, 57, 64, and 71 days after sowing.The progress and degree of disease severity were evaluated according to the following scale (Sousa et al., 2003): grade 1 (= 1 to 25%), grade 2 (= 26 to 50%), grade 3 (= 51 to 75%), grade 4 (= 76 to 100% of leaf tissue injured).From the seedling transplant period until the last severity assessment, the average temperature was 21.7 °C (range from 19.5 to 24.3 °C); the average accumulated precipitation was 86.0 mm (range from 40.0 to 148.0 mm) and the average relative humidity was 73.43% (range from 54.2 to 82.9%) (Supplementary Fig. 1).
Field-selection of a subset of lettuce accessions for subsequent greenhouse assays aiming to assess the Septoria lactucae resistance stability From the initial screening in the field, a subset of accessions classified as promising sources of resistance was selected.The cultivars 'BRS Mediterrânea' and 'Romana LNS' (identified as being tolerant accessions under commercial field conditions) were also included in these subsequent assays.Seeds of the nine field-selected accessions/cultivars ('Vera', 'Vanda', 'Romana Lente New Selection', 'Rubi', 'Isabela', 'Elisa', 'Banchu New Red Fire', 'BRS Mediterrânea', and 'Veneranda') were sowed in styrofoam trays of 126 cells filled with sterilized substrate (Plantmax ® ) and kept in a greenhouse.Seedlings (15 days after sowing) were removed from the cells and then transplanted into plastic pots (1.0 L) containing a previously sterilized mixture.The mixture was composed of 110 g of ammonium sulfate, 510 g of super simple fertilizer, 200 g of limestone, 20 L of raw rice straw, 20 L of carbonized rice straw and 40 L of cattle manure for every 200 L of soil.
The isolates were cultivated in 9 cm diameter Petri dishes containing oatmeal and agar culture medium (60 g of oat flour +18 g of agar +1000 mL of distilled water) in an incubator type BOD at constant temperature of 17 °C (12 hours light and 12 hours dark) for 13 days until sporulation (Dhingra & Sinclair, 1995).For inoculum production (conidial suspension), 10 mL of sterile distilled water was added to each plate.The conidia were released into the suspension using a soft bristle brush and the spore suspension was subsequently filtered through a double layer of gauze.The spore concentration was estimated by counting under an optical microscope using a Neubauer chamber.The suspension was adjusted to a concentration of 2 × 10 5 conidia/ mL.In the final step, 1 mL of Tween 20 ® per liter of water, was added to the suspension aiming to increase spore adhesion to inoculated leaves.

Greenhouse assay for Septoria lactucae reaction of field-selected lettuce accessions at the early vegetative phenological stage
The experiment was carried out during the months of October and November 2021.The experiment was installed in a completely randomized design in a 9 × 4 factorial arrangement (nine accessions x four isolates) with four replications and each replication constituted a pot with two plants each.The cultivar 'Rubi' served as a susceptible control.One hour before inoculation, the 1 L-pots containing the lettuce plants were irrigated up to maximum soil saturation.Then, the plants (27 days of sowing) were inoculated by spraying the leaves until the beginning of the suspension run-off.After inoculation, the plants were kept in a humid chamber (23 °C ± 2 °C) for 48 hours.Control plants were sprayed only with sterile distilled water plus Tween 20 ® .In order to avoid cross-contamination between isolates, all plants treated with different isolates were kept in different benches (at least one meter apart).Disease incidence assessments were performed by counting the number of leaves with symptoms at 11 days after inoculation and the severity at 11, 13, 19 and 23 days after inoculation, using a scale of scores as a function of the degree of visual leaf damage (Sousa et al., 2003).
Vol:. ( 1234567890) Greenhouse assay for Septoria lactucae reaction of field-selected lettuce accessions at the vegetative-reproductive phenological stage In May-June, 2022, a third bioassay was carried out with older lettuce plants of the same subset of accessions that were inoculated in order to verify the reaction to fungus S. lactucae in the transition of the vegetative to the early reproductive phenological stage.This assay was carried out with identical methodological approaches of the first greenhouse assay with exception that the plants were inoculated at the age of 50-days-old.Evaluations were performed by visual analysis of the severity of leaf damage induced by the pathogen at 57, 64, and 71 days after sowing.The progress and degree of disease severity were evaluated according to the scale of Sousa et al. (2003).

Statistical analyses for all assays
The Area Under the Disease Progress Curve (AUDPC) was calculated from the pathogen reaction data (= average severity grades) of each lettuce accession expressed by the arithmetic mean of the scores of all evaluated plants.AUDPC was calculated using the following formula: AUDPC = Σ [((y 1 + y 2 ) /2) *(t 2 -t 1 )], where y 1 and y 2 are two consecutive evaluations performed at times t 1 and t 2 , respectively (Campbell & Madden, 1990).After obtaining the severity grade values for each lettuce accession, the data were submitted to analysis of variance and the means were grouped by the Scott-Knott test (p ≤ 0.05) with the assistance of the SISVAR statistical program (Ferreira, 2011).Aiming to verify the uniformity of the disease distribution in the field trial a heat map was constructed.Pearson correlations between the two greenhouse trails was also calculated.Both analysis were done in the R softwere.

Results
In the present study, 25 out of the 42 accessions under evaluation were classified as the 'Crispy loose-leaf' (with green and purple foliage), six as 'Iceberg', four as 'Cos/Romaine', four as 'Butterhead' and three as 'Loose-leaf super crispy' ('Mimosa') morphotypes (Fig. 1).A significant effect was observed among lettuce accessions for the levels of resistance and susceptibility to S. lactucae (Table 1).The coefficient of variation for the "disease severity" variable was 14.4%, indicating adequate experimental precision.The uniformity of the disease in this assay was confirmed by the heat map (Fig. 2).Despite the gradient of phenotypes, according to the Scott-Knott test (p < 0.05) the lettuce accessions were divided into two large reaction groups based on the comparison of AUDPC means.However, none of the accessions showed an immune-like reaction.Among the evaluated accessions, 21 displayed superior levels of resistance to S. lactucae with mean AUDPC values ranging from 46.00 to 64.50 (Table 1), encompassing seven accessions of the 'Crispy green loose-leaf', eight of the 'Crispy purple loose-leaf', two of the 'Cos/Romaine', two of the 'Mimosa', one accession of the 'Butterhead', and one of the 'Iceberg' morphotype (Fig. 1).In the field assay, 21 accessions showed a reaction classified as susceptibility with mean AUDPC values ranging from 70.50 to 97.00 (Table 1).Of the group of susceptible accessions, nine were of the 'Crispy green loose-leaf, one of the 'Crispy purple loose-leaf', five of the 'Iceberg', two of the 'Cos/Romaine', three of the 'Butterhead' and one of the 'Mimosa' morphotype (Fig. 1).
In this bioassay, seven cultivars ('Vera', 'Vanda', 'Rubi', 'Isabela', 'Elisa', 'Banchu NRF', and 'Veneranda') belonging to the subgroup of accessions with the best performance under field conditions were reevaluated under greenhouse conditions (Table 2).The cultivars 'BRS Mediterrânea' and 'Romana LNS' (both displaying high levels of resistance to S. lactucae under commercial conditions) were also included in these trials.The expression of the typical disease symptoms started at the third day after inoculation in all accessions, being characterized by necrotic lesions in the leaf blade and by irregular and chlorotic areas delimited by veins.Significant differences (P ≤ 0.05) were observed among the cultivars in relation to the AUDPC values.The coefficient of variation for the variable "disease severity" was 18.22%, indicating good experimental precision.Based on the values of this index, the cultivar 'BRS Mediterrânea', displayed a superior performance with significantly lower AUDPC values (12.28) than the remaining cultivars, indicating lower levels of severity/higher level of disease resistance in this cultivar (Table 2).The accessions 'Elisa', 'Romana LNS', 'Vera', 'Veneranda', 'Vanda', 'Banchu NRF', and 'Isabela' displayed an intermediate reaction, with mean AUDPC values ranging from 13.66 to 17.63.The cultivar 'Rubi' (used as susceptible control), was moderately susceptible to the pathogen (AUDPC value = 23.72),but in significant contrast to the other cultivars.No significant differences (p ≤ 0.05) were observed in aggressiveness among the S. lactucae isolates (Table 2), exhibiting similar levels of infected leaf tissue.
The disease was much more severe when the plants were inoculated at 51 days compared to those inoculated at 27 days (Fig. 3), confirming that lettuce plants become more susceptible to the pathogen as they age (Blancard et al., 2006).In general, the accessions showed statistically significant differences (p ≤ 0.05) for the reaction to S. lactucae during the transition from the vegetative to the reproductive phenological phase (Table 3).The experimental coefficient of variation for the variable "disease severity" was 15.48%, indicating good experimental precision.The cultivar 'BRS Mediterrânea' was statistically different from the other cultivars/accessions evaluated, displaying higher levels of rate-reducing resistance with an average AUDPC of 17.09 versus 30.66 of AUDPC of the cultivar 'Rubi' (susceptible control).The cultivars 'Vanda', 'Veneranda', 'Vera',   2 and 3) when compared with trials conducted under field conditions during the rainy season (Table 1).
In both greenhouse assays, all isolates were pathogenic to all cultivars, indicating a lack of cultivarspecificity of the tested isolates (Table 3).However, there was differential interactions between some accessions and pathogen isolates.In the first greenhouse bioassay (inoculation at 27 days), the cultivar Banchu NRF was more resistant to isolates Sep-13 and Sep-23, while the cultivar Isabela was more resistant to the isolates Sep-34 and Sep-39, and the cultivar Rubi to the isolate Sep-26.In the second greenhouse bioassay (inoculation at 51 days) four cultivars presented differential interaction with the pathogen isolates.The cultivar BRS Meditrrânea, beside been the most resistant, was more susceptible to Sep-26 isolate than the other isolates.The cultivar Veneranda, beside presented high resistance, was less resistant to the isolate Sep-34.The cultivar Vera was more resistant to the isolate Sep-26 them than to other isolates.The cultivar Isabela was more susceptible to the isolate Sep-34 them than to other isolates.There was significative correlation between the two greenhouse assays (p = 0,01) but very low (cor = 0.28).

Discussion
Septoria leaf spot is considered one of the main fungal diseases in lettuce in Brazil.From an epidemiological point of view, its incidence and severity are favored by conditions of high humidity with temperatures ranging between 20 and 24 °C.The presence of dew and high relative humidity allow the prompt germination of S. lactucae conidia that can subsequently penetrate into the plant tissue, inducing  the characteristic set of symptoms (Bedlan, 1999;Blancard et al., 2006;Nao, 2008).The levels of disease observed under field conditions in the present investigation corroborate the climatic condictions, such as high relative humidity and rainfall, favoring epidemiological parameters indicated as favorable to S. lactucae (Blancard et al., 2006).The assay was conducted coinciding with the months of January to March, when high levels of rainfall are observed in Central Brazil (Blancard et al., 2006).The weekly averages of temperature (21.7 °C), precipitation (86.0 mm) and relative humidity (73.43%) during the field assay are within the appropriate range for disease onset and progression, providing ideal conditions for the development of the pathogen.The heat map shows that the disease was well distributed in the field trial allowing a good discrimination of the reactions of the cultivars.In the field trial, it was possible to separate the accessions into two discrete reaction groups based upon the comparison of their AUDPC means.No immunity-like source of resistance was detected in the cusltivars evaluates, as observed before by Sousa et al. (2003).However 21 accessions were identified displaying higher levels of rate-reducing field resistance to S. lactucae with significant lower AUDPC values, which is the major indicator of plant accessions with improved levels of quantitative or field resistance (Vanderplank, 1984;Campbell & Madden, 1990).In this trial, a better performance was observed, as a group, of the cultivars of the 'Crispy loose-leaf'.The top ten cultivars displayed a majority of accessions from this morphotype.Significant differences were also observed under greenhouse conditions among seven field-selected accessions ('Vera', 'Vanda', 'Rubi', 'Isabela', 'Elisa', 'Banchu NRF' and 'Veneranda') as well as the cultivars 'BRS Mediterrânea' and 'Romana LNS' for the response to four S. lactucae isolates.Again, two cultivars of the 'Crispy loose-leaf' group ('BRS Mediterrânea' and 'Vanda') displayed superior levels of stable performance against this collection of fungal isolates in both phenological stages.
Septoria leaf spot in lettuce might be characterized as a typical "low-sugar disease" (sensu Vanderplank, 1984), becoming more severe in the transition from the vegetative to the reproductive phenological phase.In general, the field-selected lettuce accessions displayed statistically significant differences among themselves for the reaction to S. lactucae in the transition from the vegetative to the reproductive phase, with special emphasis for the cultivars 'BRS Mediterrânea' and 'Vanda' that displayed significantly lower AUDPC values.Ratereducing resistance is a complex phenotype, resulting from the coordinated action of different defense mechanisms, either impairing or avoiding the plant invasion and/or damage induced by variants of a given pathogen species (Heath, 2000).The major phenotype associated with quantitative disease resistance in different pathosystems is the ability of a subset of host accessions in reducing the rate of disease progress, which can be demonstrated by significantly lower AUDPC values (Willocquet et al., 2017).In general, quantitative/rate-reducing resistance is based on the presence of biochemical barriers as well as distinct physical, anatomical, and morphological mechanisms (Dixon, 2001), which were not yet evaluated in the lettuce x S. lactucae pathosystem.
It was observed few differential reactions between the pathogen isolates and the lettuce cultivars.The low variability, in terms of aggressiveness/virulence, observed among our S. lactucae isolates can be explained by the previous observations that the genetic diversity of these fungal populations is extremely low under Brazilian conditions (Cabral et al., 2022).Probably one or a few isolates of the pathogen were introduced in the country and they are being disseminated in the different lettuce-producing regions, via contaminated seeds, resulting in not yet significant variations for the aggressiveness profile within these S. lactucae populations.In addition, most lettuce genotypes evaluated here are commercial cultivars, that were selected in many self-fertilization cicles, therefore presenting very low genotypic and fenotypic variability.
The results from our study indicate that there are distinguishable levels of rate-reducing resistance across the different morphotypes of lettuce with a higher frequency of promising accessions within the 'Crispy green loose-leaf' group.Brazil is one of the few countries in the world where the 'Crispy green loose-leaf' lettuce segment is the market leader, with the release in recent years of ≈ 380 new cultivars, with 70% of them belonging to this morphotype.Thus, it is possible to speculate that the wide acceptance (especially by the growers) of cultivars from this morphotype might be associated with their superior adaptation traits to Brazilian Neotropical regions, including their higher levels of rate-reducing resistance to foliar pathogens such as S. lactucae.
It is important to highlight that accessions of different lettuce morphotypes were also detected showing promising levels of rate-reducing resistance to S. lactucae and they can be incorporated in breeding programs aiming to broaden the genetic basis of resistance.The identification of genetic factors controlling resistance to S. lactucae is crucial since the development of cultivars with characteristics of tolerance and/or resistance to this pathogen is an important strategy to guarantee the sustainability of the production system of this vegetable across Neotropical regions.

Fig. 1
Fig. 1 Heat map showing the space/temporal intensity of the disease in the field trial

Fig. 2
Fig. 2 Classes of reaction (resistance or susceptibility) to Septoria lactucae of 42 cultivars/accessions of different lettuce morphotypes evaluated under open-field conditions (natural inoculum) in Brasilia-DF, Brazil.Codification of the lettuce

Fig. 3
Fig. 3 Average disease severity on the first (27 days) and second (51 days) greenhouse assays for resistance of nine lettuce cultivars to four Septoria lactucae isolates

Table 1
Mean values for the Area Under the Disease

Table 2
Mean values of the Area Under Disease Progress Curve (AUDPC) observed in a greenhouse assessment of the severity of four Septoria lactucae isolates in a subgroup of *Lettuce morphotypes: 'Crispy Green Loose-Leaf' (CGL), 'Crispy Purple Loose-Leaf' (CPL), 'Iceberg' (ICE), 'Cos/Romaine' (ROM), 'Butterhead' (BHD), and 'Mimosa' (MIM) **Means followed by distinct lowercase letters in the column and appercase letters in the row differ from each other, by the Scott-

Table 3
Mean values of the Area Under Disease Progress