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

Abstract

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.

Introduction

Lettuce (Lactuca sativa L.; Asteraceae family) is among the most economically important vegetable crops around the world (Shatilov et al., 2019). In Brazil, the area employed for lettuce cultivation has continually grown, encompassing ≈ 87,000 hectares. The annual production is around 1.5 million tons, involving ≈ 670,000 farmers (Pessoa & Machado Júnior, 2021). The Brazilian market is diversified, including distinct lettuce morphotypes, such as ‘Butterhead’, ‘Crispy green loose-leaf’, ‘Iceberg’, ‘Batavian’, ‘Loose-leaf super crispy’ (= ‘Mimosa’), ‘Cos/Romaine’, and ‘Crispy purple loose-leaf’ segments (Sala & Costa, 2012). The ‘Crispy green loose-leaf’ is the leading morphotype, occupying around 53% of the total market share (Pessoa & Machado, Júnior 2021).

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 in 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 their 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.

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/tolerance in a genetically diverse lettuce germplasm collection.

Material And Methods

Fungal isolates and inoculum production for the subsequent greenhouse bioassays

Four S. lactucae isolates were employed in the bioassays viz. Sep13 (063.482 TRA), Sep26 (063.490 TRA), Sep34 (063.496 TRA) and Sep39 (063.501 TRA). All these isolates were classified as S. lactucae via molecular analyses (Cabral et al., 2022). The isolates were cultivated in 9 cm diameter Petri dishes containing oatmeal and agar culture medium (60 grams of oat flour + 18 grams of agar + 1000 mL of distilled water) in an incubator type BOD at constant temperature of 17 ^oC (12 hours light and 12 hours dark) for 13 days until sporulation (Dhingra and Sinclair 1995) for each isolate. For inoculum production (= conidial suspension for each isolate), 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 x 10⁵ 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. One hour before inoculation, the 1L-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 (23ºC ± 2ºC) 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 (= severity). The experiment was carried out in a completely randomized design in a 9 x 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.

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 following 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 [((y₁ + y₂) /2) *(t₂ - t₁)], where y₁ and y₂ are two consecutive evaluations performed at times t₁ and t₂, respectively. 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).

Results

In the present work, 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. In general, the lettuce accessions were divided into two large reaction groups based on the comparison of AUDPC means using the Scott-Knott test (p < 0.05). 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 re-evaluated 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. All isolates were pathogenic to all cultivars, indicating a lack of cultivar-specificity of the tested isolates (Table 3).

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 levels of 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’, and ‘Romana LNS’ displayed intermediate values of AUDPC (ranging from 18.91 to 22.2), while a higher disease severity was observed for the cultivars ‘Banchu NRF’ (AUDPC: 24.97) followed by ‘Isabela’ (AUDPC: 25.50) and ‘Elisa’ (AUDPC: 28.13). However, we observed overall lower AUDPC values and lower levels of aggressiveness of S. lactucae isolates under greenhouse conditions (Tables 2 and 3) when compared with trials conducted under field conditions during the rainy season (Table 1).

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 20–24°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 epidemiological parameters indicated as favorable to S. lactucae. The assay was conducted coinciding with the months of January to March, when high levels of rainfall are observed in Central Brazil. 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.

In the field trial, it was possible to separate the accessions into two discrete reaction groups based upon the comparison of their AUDPC means. Although no immunity-like source was detected, 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 phonological 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. Rate-reducing 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.

The lack of variability, in terms of aggressiveness, 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 no yet significant variations for the aggressiveness profile within these S. lactucae populations.

Although no immunity-like source was detected, a subset of lettuce accessions displayed higher levels of resistance of the rate-reducing resistance to S. lactucae. Our results 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.

Declarations

Authors’ Contribution

David Nataren Perdomo contributed to the study design, performed the experiments and worote the manuscript. Leonardo Silva Boiteux contributed to the study design, co-developed the idea and wrote the manuscript. Ailton Reis contributed to the study design, co-developed the idea and wrote the manuscript. Tiago Bezerra Torres performed the experiments, commented and approved the final version of the manuscript. Cleberly Evangelista Santos performed the experiments, commented and approved the final version of the manuscript. Cléia Santos Cabral performed the experiments, commented and approved the final version of the manuscript.

Conflicts of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Consent for publication

All authors agree with the publication of the work in Phytoparsitica.

Availability of data and material

The data and material that support the findings of this study are available from the corresponding author upon reasonable request.

Funding

Not applicable

Acknowledgments

The authors thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the support to the Postgraduate Program in Plant Pathology of the Federal Rural University of Pernambuco – UFRPE. Ailton Reis and Leonardo S. Boiteux thank the CNPq for their scholarship grants.

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