Effect of abiotic stress on the endophytic fungal community in Brassica napus L. (winter canola) leaves

Effect of abiotic stress on the endophytic fungal community in Brassica napus L. (winter canola) leaves | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of abiotic stress on the endophytic fungal community in Brassica napus L. (winter canola) leaves Rocio Florencia Gaiser, Vanesa Eleonora Tossi, Axel Joel Rizzo, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6708525/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Winter canola is an oleaginous plant whose seeds are used in the food and biodiesel industries. Fungal endophytes are found in asymptomatic tissues of almost all plant lineages, promoting positive responses to stress situations. This study aims to characterize the fungal endophytic community of canola leaves (var. Hornet) grown under abiotic stress, comparing them with those plants with no apparent stress signs. Our results showed that canola has a community of endophytic fungi distributed in the Phylum Ascomycota, with members of Alternaria and Stemphylium as dominant groups. We found 231 endophytic fungi strains, belonging to 2 genera and 29 fungal morphotypes. A greater diversity and richness of endophytic fungi were found in canola populations grown on saline soil (14 species, SSP) and waterlogged soil (11 species, WSP). Conversely, the canola population with no apparent abiotic stress (NASP) had only 3 endophytic species. The highest estimated species richness was found in the SSP (17.2), followed by the WSP (12.6), and the NASP (3.4). We also observed NASP showed very low endophyte colonization compared to stressed plants, being 6 and 4 times higher on WSP and SSP, respectively. Additionally, plant parameter analyses showed that canola leaves of SSP and WSP accumulated more water than canola leaves of NASP. The results obtained contribute to the knowledge on the dynamics of the endophytic fungal community associated with canola cultivation in the field, allow estimation of mycodiversity, and might be useful in research aimed at crop growth promotion. Winter canola Alternaria Stemphylium abiotic stress fungal endophytes Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Fungal endophytes are microorganisms that infect healthy tissues of all plant lineages without causing any symptoms of disease (Arnold et al. 2007 ; Suryanarayanan 2013 ). Horizontally transmitted endophytes are very diverse and mostly belong to the Phylum Ascomycota (Arnold and Lutzoni 2007 ). Endophytes can have a significant impact on plant communities by conferring tolerance to biotic and abiotic stress, increasing biomass and reducing water consumption, or decreasing fitness by altering resource allocation (Dickinson et al. 2021 ; Kumar and Nautiyal 2022 ; Rodriguez et al. 2009 ). Previous studies (Fadiji and Babalola 2020a , b ; Grabka et al. 2022 ) even suggest that endophytes may contribute to the establishment of plant species by enhancing their competitiveness.These reports agree that these organisms play a fundamental role in plant responses to various external disturbances, which are significant at the ecological level. Current knowledge about plant adaptation to environmental changes not only suggests that plants can adapt, but also that they can interact, modify and select specific microbial communities to mitigate the negative effect of different stressors (Rodriguez and Durán 2020 ). This protective function appears not to be carried out by all microorganisms, but rather by some microbial groups (Rodriguez and Durán 2020 ). Considering the increasing number of studies supporting the idea that plants selectively interact with their surrounding microbiome (Rodriguez and Durán 2020 ; Suryanarayanan and Shaanker 2021 ), it becomes evident that the study of endophytic communities is of utmost importance to build a comprehensive map of interactions and their effects for species of interest. Brassica napus L. (also known as rape, rapeseed and canola) is one of the most important oilseed crops worldwide, since the oil from its seeds is used in food industry and for biodiesel production, being also widely used in crop rotation and forage (Gómez et al. 2018 ; Card et al. 2015 , among others). Traditional rapeseed was found harmful for the animals that graze on it, since it caused myocardial alterations due to the presence of erucic acid. For this reason, the oil was destined for industrial purposes. It was not until 1970 that Canada and Europe achieved varieties improved, with low content of erucic acid and glucosinolates in the seeds. These varieties are called “00” or “double zero” in Europe and CANOLA (CANadian Oil Low Acid) in Canada. The production of rapeseed in the world was recorded at 87.2 million tons in the year 2022, while the record of the yield of rapeseed in the world is 21,824 (100g/ha) (FAO, 2022). In 2022, the continent with the highest production and yield of rapeseed was Europe, with 29.7 million tons and 29,197 (100g/ha) respectively. The second continent with the highest production was Asia, with 29.1 million tons of production and 17758 (100g/ha) of yield, and in the third place was America, with a production of 21.4 million tons and a yield of 21339 (100g/ha) (FAO, 2022). Canada is the world's leading country producer of canola, with a production of 18.7 million tons and a yield of 21,747 (100g/ha) (FAO, 2022). In Argentina, these varieties (“00” or CANOLA) introduced from Europe and Australia are currently used (Sánchez-Vallduví and Chamorro 2023 ). The incorporation of this crop into Argentinian production schemes presents numerous advantages considering the improvements in genetics and management over the last decade, which have raised the average crop yields in the country to 1,800 kg/ha, compared to the historical 1,400 kg/ha (Sánchez-Vallduví and Chamorro 2023 ; Schwab 2010 ). According to FAO data (2022), the yield of canola was 20,482 (100g/ha) and production was 52,915 tons in 2022 for Argentina. Winter canola is an important alternative in agriculture and the oil industry in Argentina, since it supplies oil in the summer season and promotes rotation with other crops such as winter cereals (wheat, barley, oats) and does not compete in the oil industry with soybeans and sunflowers (Kirkegaard et al. 2021 ). Despite these advantages, canola production has not reached a stable volume due to technological limitations regarding the improvement and management of the crop (de Emilio 2017 ; Iriarte and López 2014 ). One of the factors that negatively affects canola crop is the abiotic salinity stress. The yield of many brassica species, such as canola ( Brassica napus ), mustard ( Brassica juncea L.), cabbage ( Brassica oleracea L.) and turnip ( Brassica rapa L.), are highly affected by salinity (Jan et al. 2016b ). It has been previously reported that salt stress causes inhibition of canola growth and development (Akhter et al. 2023 ; Bacarin et al. 2011 ). The aim of this work is to characterize the fungal endophytic community of canola leaves (var. Hornet) grown under different field conditions (saline soil and waterlogged soil). This work also explores the relationships between plant parameters, soil parameters and endophytic diversity. The characterization of foliar endophytic communities associated with a crop such as canola is of great interest, promoting new technological approaches to crop stress tolerance through biologically active compounds and complexes and the study of their physiological effects. To our knowledge, the study by Zhang et al. ( 2014 ) is the only one that studies the endophytic community of B. napus grown in fields in China (leaves, stems and roots). Materials and Methods Site and soil characterization The study was carried out in three canola populations located in a 35 ha. field in General Juan Madariaga, Province of Buenos Aires, Argentina (37° 01' 41.9" S 57° 07' 56.7" W). The soil was differentiated by three field conditions: no apparent abiotic stress (NAS), a saline condition according to local considerations and a frequently waterlogged condition (referred to as SS and WS, respectively). In each of these soil conditions different populations of canola developed and were subsequently analyzed (referred to as NASP, SSP and WSP, respectively). Samples were taken from different zones under different conditions (NAS, SS and WS). Composite samples of each soil from 3 sampled zones were placed in bags. The 3 soils were characterized through analytical tests conducted at the Soil, Water, and Plant Laboratory, Soil Science Department, Faculty of Agronomy, University of Buenos Aires. Sampling and plant material The studies were carried out on 3 populations of canola, variety Hornet (Al High Tech), planted in each of the previously described soils. The plants were sown simultaneously on April 13th, 2019, by the agribusiness Hórreos del Sudeste S.A. Sampling was conducted 140 days after planting. Within each population (according to field conditions), plants were randomly selected, and fully expanded healthy young leaves were chosen. Four leaves per plant were sampled from 40 plants of each population, resulting in a total of 480 leaves selected from 120 healthy plants (without visible alterations or damage to the aerial part). Plant parameters To calculate biomass, the largest leaf of the four leaves per plant was used. Biomass in canola leaves was estimated as the dry weight of whole leaves and as the dry weight of 7 mm diameter discs for each population. For water content, the same leaves used to calculate biomass were utilized. The water content of the canola leaves was calculated as a percentage relative to the fresh weight of the leaves: $$\:WC\:=\:\frac{FW\:-\:DW}{FW}\:*\:100\%$$ Where: WC = water content; FW = fresh weight; DW = dry weight. A precision balance (Ohaus Traveler TA302, China) was used to obtain whole leaf fresh and dry weight and calculate their water content. The chlorophyll content was measured in one of the four sampled leaves per plant. Chlorophyll a (Chl a ) and chlorophyll b (Chl b ) were measured by immersing 7 mm diameter leaf discs in 1 mL of N-N dimethylformamide (DMF) solution. The discs were previously weighed on a precision balance (Ohaus Explorer, USA). They were then incubated in the dark at 4°C, and after 7 days, the absorbance of the solution was measured at 663.8 nm and 646.8 nm using a spectrophotometer (Shimadzu UV-1800, Kyoto, Japan) with quartz cuvettes. The concentration of chlorophyll was calculated in µg/mL according to the following equations (Porra 1989, 2002 ): $$\:\left[Chl\:a\right]\:=\:12{{A}^{663.8}\:-\:3.11A}^{646.8}$$ $$\:\left[Chl\:b\right]\:=\:20.78{{A}^{646.8}\:-\:4.88A}^{663.8}$$ $$\:[Chl\:a+b]\:=\:17.67{{A}^{646.8}\:+\:7.12A}^{663.8}$$ The chlorophyll concentration was normalized to the DMF volume and to the weight of the discs (µg/mg of tissue, with tissue as the fresh weight of 7 mm diameter discs). Isolation and morphological identification of endophytic fungi The leaves of the 120 selected plants were processed as follows: 1 leaf per plant was used to isolate fungal endophytes, recovering strains between 24 and 86 hours after sowing, to characterize the fungal communities. The leaf fragment, was superficially sterilized with 70% ethanol for 1 min, 4% Sodium Hypochlorite for 2 min, 50% ethanol 30 s and 3 washes with sterile distilled water for 2 min each, then the leaf fragment was left to dry on sterile paper before sowing it in Petri dishes. Isolates were obtained from 14–15 fragments per leaf of 5 mm diameter in Petri dishes of 9 cm-diameter with 2% Malt Extract Agar with chloramphenicol (100 mg L − 1) (2% MEAc) cultured at 23–25°C in the dark. All strains were studied under an optical microscope (Olympus BX41, Tokyo, Japan) with an attached Infinity1 camera. The macromorphological characteristics of the colonies were also evaluated: color and texture of the mycelium, reverse of the culture, reproductive structures, edge of growth, among others. Taking these characteristics into consideration, the strains were grouped in morphotypes. DNA extraction, PCR and sequencing Twenty-nine representative strains of the most abundant morphotypes (morphotypes representing 95% of the obtained strains) were selected for DNA sequencing studies. These selected strains were cultured on 2% MEA and the cultures were incubated at 23–25°C for 10–21 days in the dark. Genomic DNA was extracted from fresh mycelial culture using the DNeasy UltraClean microbial kit (Qiagen, Germany), following the manufacturer's protocol. The ITS (Internal Transcribed Spacer) of the rRNA gene was amplified using the universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGTCTATTGATATGC-3') under the following PCR conditions: initial denaturation at 94°C for 3 min, 45 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 1 min, and a final extension at 72°C for 10 min. The PCR reactions were carried out in a final volume of 50 µL, containing 5 µL of 10x buffer, 0.2 mM of each dNTP, 0.4 µM of each primer, 0.5 µL of Taq DNA polymerase (Qiagen, Germany), 5 µL of fungal genomic DNA, and 36.5 µL of milliQ water. The RPB2 region (second largest subunit of RNA polymerase) was amplified using the primers RPB2–5F2 (5'-GGGGWGAYCAGAAGAAGGC-3') and RPB2–7cR (5'-CCCATRGCTTGTYYRCCCAT-3') under the following PCR conditions: 5 cycles of 45 s at 94°C, 45 s at 60°C, and 2 min at 72°C, followed by 5 cycles with a hybridization temperature of 58°C, followed by 30 cycles with a hybridization temperature of 54°C, and a final extension at 72°C for 7 min. The PCR reactions were carried out in a final volume of 50 µL, containing 5 µL of 10x buffer, 3 µL of Mg, 0.2 mM of each dNTP, 0.4 µM of each primer, 0.5 µL of Taq DNA polymerase (INBIO Highway, Argentina), 2 µL of fungal genomic DNA, and 36.5 µL of milliQ water. All PCR products were purified using the DNA PuriPrep-GP kit (INBIO HighWay, Argentina). All DNA fragments were sequenced by Macrogen Inc sequencing service (South Korea). Phylogenetic analyses The obtained sequences were initially compared with sequences from the NCBI database (GenBank) using BLAST (Basic Local Alignment Search Tools) to search for highly similar sequences as the first step for subsequent phylogenetic studies (Supplementary material S1 to S12). The names of the species to which the reference sequences belong were maintained as they appear in GenBank. The DNA sequences were edited and manipulated using the BioEdit Sequence Alignment Editor program (Hall, 1999 ) and aligned with the MEGA-X program (Kumar et al. 2018 ) using the CLUSTAL option, leaving all parameters at their default values. Maximum parsimony (MP) trees were constructed using the MEGA-X program, with 1000 bootstrap replicates, and gaps were treated with 95% partial deletion. The evolutionary model selection and maximum likelihood (ML) phylogenetic analyses were conducted in IQ-TREE v. 2.2.0 (Minh et al. 2020 ). The most appropriate model, according to Bayesian Information Criteria (BIC), was selected by the ModelFinder algorithm (Kalyaanamoorthy et al. 2017 ) included in IQ-TREE. Support for internal nodes was estimated using 1000 ultrafast bootstrap replicates (UFboot; Hoang et al. 2018 ) and the Shimodaira-Hasegawa approximate likelihood ratio test (SH-aLRT) with 1000 replicates. The command line used for the pre-aligned ITS1 and RPB2-5f matrices was: iqtree2 -s inputfile.nex -bb 1000 -alrt 1000 –nt AUTO. According to BIC, the best-fitting substitution model selected for Alternaria ITS was SYM + I + G4 (i.e., symmetrical model, a proportion of invariable sites [I = 0.532], and a Gamma distribution with four categories [alpha parameter = 0.5]); the best-fitting substitution model selected for Stemphylium ITS was TIM2e + G4 (i.e., transition model, equal base frequency, and a Gamma distribution with four categories [alpha parameter = 0.275]), and the best-fitting substitution model selected for Alternaria RPB2 was TIM2e + I + G4 (i.e., transition model, equal base frequency, a proportion of invariable sites [I = 0.513], and a Gamma distribution with four categories [alpha parameter = 1.05]). The trees were visualized using FigTree v.1.4.4 (Rambaut 2018), with branch lengths measured in the number of substitutions per site. Bayesian inference was conducted using MrBayes v. 3.2.7a program (Ronquist et al. 2012 ), with 5,000,000 generations, a chain temperature value set at 0.05, and a burn-in set at 25%. Statistical analysis Species accumulation curves and estimates of total fungal endophyte richness were inferred using EstimateS version 9.1.0 (Colwell 2013 ). Endophyte diversity was measured by the Shannon diversity index (H) using the same software. The similarity between the endophyte communities studied was evaluated with the Sørensen similarity index using the following formula: $$\:S\:=\frac{2C}{A\:+\:B}$$ Where S is the degree of similarity, A and B are the number of endophytic species from two of the field conditions (NAS, SS, or WS) and C is the number of endophytic species common to both field conditions. To assess the influence of environmental variables (different field conditions) on the presence and frequency of endophytes (biodiversity of the foliar fungal endophyte community), a canonical correspondence analysis (CCA) was performed using R Core Team (2020). Hellinger transformation was conducted to correct potential statistical errors associated with rare species (Legendre & Gallagher 2001 ). Statistical analyses were performed in R version 4.0.3 (R Core Team, 2020). A one-factor ANOVA was conducted (alpha < 0.05). The plant parameters included leaf biomass, leaf disc biomass, leaf water content, chlorophyll a , chlorophyll b , and total chlorophyll (chlorophyll a + b ). The factor analyzed was field conditions (NAS, SS, and WS). Variance was modeled using a Generalized Least Squares (GLS) linear model, and maximum likelihood estimations were performed in cases where the assumption of homoscedasticity was not met (leaf biomass, leaf disc water content). The best model was chosen based on the Akaike Information Criterion (AIC). Pairwise comparisons were conducted using Tukey's Honestly Significant Difference (HSD) test. Results Identification of endophytic fungi In this study, 1787 leaf fragments were evaluated, obtaining a total of 231 strains, belonging to 3 genera and 8 species. A total of 21 strains were obtained from NASP leaves, 87 strains from SSP leaves, and 123 strains from WSP leaves. The genera Stemphylium Wallr. ( Pleosporaceae, Ascomycota ) and Alternaria Nees. ( Pleosporaceae, Ascomycota ) were the most abundantly represented, accounting for 95% of the strains (Table 1). In addition, Fig. 1 shows the distribution of taxa in each field condition. Table 1 Number, relative frequencies and sample type of isolated strains of the three field conditions of canola populations: without abiotic stress canola populations (NASP), saline soil canola populations (SSP) and waterlogged soil canola populations (WSP). Other taxa correspond to a total of 9 morphotypes. In the first column Genbank accession numbers of each strain are given between parentheses. Taxon (strain GenBank accession number) Number of strains Relative frequency NASP SSP WSP Alternaria sp.1 (ITS/RPB2: PP949836/-, PP949839/-, PP949840/PP960212, PP949842/PP960214) 0 4 13 7.4 Alternaria sp.2 (ITS: PP949838/PP960211, -/PP960217) 0 2 14 6.9 Alternaria sp.3 (ITS/RPB2: PP949841/PP960213, PP949843/PP960215, PP949844/PP960216) 1 4 3 3.5 Alternaria sp.4 (ITS/RPB2: PP949837/PP960210) 0 1 0 2.3 Stemphylium sp.1 (PP950360, PP950361, PP950364, PP950365, PP950370, PP950377, PP950378). 16 53 70 60.2 Stemphylium sp.2 PP950366, PP950367, PP950368, PP950371, PP950373, PP950375, PP950376) 4 10 10 10.4 Stemphylium sp.3 (PP950369, PP950372) 0 1 5 2.6 Stemphylium aff. amaranthi (PP950362, PP950363, PP950374) 0 6 2 3.5 Other taxa 0 6 6 5.2 TOTAL 21 87 123 102 In order to improve the taxonomic identifications, a total of 3 matrices with 442 sequences were analyzed (68 sequences for the ITS gene from strains assigned to Stemphylium spp., 186 sequences for the ITS gene and 188 sequences for the RPB2 gene from strains assigned to Alternaria spp.), including the 37 sequences obtained from the 29 strains obtained in this study. Characterization using molecular markers and phylogenetic analyses placed the strains recovered during this study within the genera Alternaria and Stemphylium . Phylogenetic analyses (Supplementary material S1 to S6) grouped the Alternaria strains within the section Alternaria , distinguishing the sequences obtained into 4 different species, without a clear grouping to a particular species within the section. The phylogenetic trees corresponding to the genus Alternaria with the ITS gene and analysis reveal that the section Alternaria is a sister clade of the group formed by the section Alternantherae and the section Sonchi under maximum parsimony (bootstrap support of 93%) (see Supplementary material S6); under maximum likelihood, the section Alternaria is positioned as a sister clade of the section Sonchi but without robust support values (see Supplementary material S5); under Bayesian inference, the section Alternaria is a sister clade of the section Sonchi , but without posterior probability values exceeding the minimum value to consider that clade as true under the study conditions (see Supplementary material S4). With this marker, species-level identification was not achieved for any of the strains in this study. When analyzing the phylogenetic trees obtained for the genus Alternaria with the RPB2 gene, it is observed that the section Alternaria is positioned as a sister clade of the section Alternantherae under maximum parsimony (bootstrap of 98%), maximum likelihood (SH-aLRT = 99.9% and UFB = 100%), and under Bayesian inference (posterior probability of 100%) (see Supplementary material S1, S2, and S3). The phylogenetic trees obtained for the Stemphylium genus were inconclusive, as the supports and posterior probabilities are low. Additionally, all 3 trees exhibit polytomies. However, it can be observed that strains 3, 7, and 25 group together with Stemphylium amaranthi Y.F. Pei & X.G. Zhang in all 3 topologies, and strain 2 is positioned within the clade of Stemphylium vesicarium (Wallr.) E.G. Simmons with a support of 98% under maximum parsimony (see Supplementary material S7, S8, and S9). For these results, three strains were identified as belonging to Stemphylium aff. amaranthi . Diversity and species composition The canola populations from soils with abiotic stress (SSP and WSP) exhibited the highest species richness among the studied populations, with a total of 14 species in the canola SSP and 11 species in the WSP. Meanwhile, the canola population from no apparent abiotic stress (NASP) presented 3 endophytic species. The highest estimated species richness (Bootstrap Mean) was found in the canola population from saline soil (17.2), followed by the waterlogged soil canola population (12.6), and the without abiotic stress canola population (3.4). The Shannon-Wiener index (H) gave 0.67 for NASP, 1.51 for WSP and 1.52 for SSP. Similarity between oilseed rape populations under different field conditions (Sørensen's index), gave 0.42 between NASP and SSP, 0.5 between NASP and WSP and 0.67 between SSP and WSP. The species accumulation curves of the canola populations growing in different field conditions are shown in Fig. 2. Analysis of environmental factors and endophytic fungal diversity Soil characterization The analyses of the three soils belonging to the area where canola populations were sampled (WAS, SS and WS) are shown in Supplementary material S13. The canonical correspondence analysis (CCA) was conducted to detect the variance explained by environmental variables (field conditions) in the structure of the endophytic fungal community in canola leaves in terms of the presence and frequency of endophytes. The ordination plot was generated to visualize the associations of fungal communities with environmental predictors (Fig. 3). In Fig. 3, the first and second axes explained 65% and 35% of the variance in the biodiversity of the foliar fungal endophytic community, respectively. The factors pH and EC (soil electrical conductivity) contributed most to the differences between the foliar fungal endophytic communities and the environmental factors. The first axis (CCA1) positively correlated with EC, and the second axis (CCA2) positively correlated with soil pH. Although the CCA was not statistically significant (p > 0.05), a trend can be observed indicating that there is higher richness of endophytic species on canola leaves under abiotic stress field conditions (saline and waterlogged). Plant parameters Abiotic stresses such as waterlogging and salinity negatively impact plant growth and development, affecting chlorophyll levels, as well as the production and quality of agricultural products. In this work, different plant parameters were analyzed using healthy leaf samples from different populations of canola: a) biomass in leaf (expressed as dry weight in grams); b) biomass in leaf discs (expressed as dry weight in milligrams); c) water content in whole leaves (expressed as a percentage relative to fresh weight); d) concentration of chlorophyll a ; e) concentration of chlorophyll b ; f) concentration of chlorophyll a + b (total). Fig. 4 showed the obtained data from each population. Fig. 4a shows that the weight of dry leaves from NASP is significantly higher than that of the leaves from the canola populations growing under abiotic stress (SSP and WSP) and the dry weight of whole leaves of SSP being significantly lower than WSP and NASP. The water content of whole leaves of SSP and WSP is significantly higher than the water content of leaves from NASP (Fig. 4b). Dry weight of leaf disks from canola populations under saline stress is significantly higher than dry weight of leaf disks from canola populations growing under waterlogging stress and without abiotic stress (Fig. 4c). There are no significant differences between the dry weight of leaf disks from canola populations in waterlogged soil and those from canola populations without abiotic stress. As observed in Fig. 4d and f, the concentration of chlorophyll a and total chlorophyll ( a + b ) is lower in the leaves of canola populations from saline and waterlogged soils compared to leaves from plants grown without abiotic stress. Discussion In this work, we studied the fungal endophytic community colonizing leaves of B. napus , from three populations subjected to different field conditions, by performing analyses on soil and plant parameters and their associated endophytes. The results showed the presence of two genera: Alternaria and Stemphylium . The molecular and phylogenetic analyses using molecular markers improve the identification of strains belonging to these genera. Regarding the genus Alternaria , phylogenetic topology placed the strains from this study within the section Alternaria , and the phylogenetic hypothesis suggests the presence of more than one species belonging to Alternaria . The section Alternaria appears as the sister clade of the section Alternantherae . This result is in agreement with the results reported by Lawrence et al. 2013 and Woudenberg et al. 2013 , 2014 , and 2015 . The phylogenetic trees obtained for the genus Stemphylium were inconclusive, as the supports and posterior probabilities are low. Additionally, all analyzed trees exhibit polytomies. However, it can be observed that strains 3, 7, and 25 cluster together with Stemphylium amaranthi in all topologies and therefore could be assigned to Stemphylium aff. amaranthi . Within the genus Stemphylium , 28 species can be distinguished based on regions of the ITS, GAPDH, and cmdA genes (Woudenberg et al. 2017 ). More genes and further studies are needed to improve the taxonomic placement of obtained strains. This study shows that 20.1% of the fungal strains in total canola leaves (NASP) and 12.6% of the fungal strains on the leaves of the canola population under salinity stress (SSP) belong to the genus Alternaria section Alternaria. Previously this genus had been registered as endophytes of B. napus (Zhang et al. 2014 ). Khalmuratova et al. ( 2020 ) studied the endophytic community of roots of halophytic plants, Suaeda australis Moq., Suaeda maritime (L.) Dumort., and Phragmites australis (Cav.) Trin. ex Steud., and obtained 42%, 53%, and 33% root isolates with endophytic colonization by Alternaria alternata (Fr.) Keissl., this being the most abundant endophyte in these plants. The results presented here appear to associate the presence of the genus Alternaria as predominant under abiotic stress conditions (salinity and waterlogging) and higher endophytic diversity than under no apparent abiotic stress conditions. The high diversity may be due to environmental conditions (salinity or waterlogging) playing a determining role in leaf susceptibility to endophytic organisms, as abiotic stress may be a necessary factor for establishment in the host plant. Thus, Alternaria seems to be a frequent endophyte associated with plants grown in saline stress environments (Bouzouina et al. 2020 ; Khalmuratova et al. 2020 ; Sun et al. 2011 ). This study shows that the genus Stemphylium is highly represented in the leaves of the three canola populations studied, including the canola population growing under salinity. Stemphylium contains 96 species and includes plant pathogenic, endophytic, and saprophytic fungi with worldwide distributions. Unlike Alternaria there are a few reports of this genus as endophyte, however have been isolated as endophytes from marine and terrestrial habitat. Stemphylium solani G.F. Weber was isolated as a fungal endophyte of the leaves from the medicinal plant Artemisia absinthium L. and the bioactive ethyl acetate fungal extract showed antifungal, insect antifeedant and nematicidal activity (Diaz et al. 2014 ). Goncalves et al. (2021) shows that inoculation of the salt marsh plant Salicornia sp. with a root isolate of the genus Stemphylium positively influenced total biomass production and nitrogen concentration in roots under salinity condition that was optimal for plant growth (150 mM NaCl). The data obtained in our work, in terms of the representation of Stemphylium in the populations studied, could suggest that strains belonging to this genus could be responsible for the plant responses registered in saline field conditions. One of the most interesting results obtained is the diversity (expressed as the Shannon index) index value which reaches 1.52 in saline soils, which is much more diverse than the endophytic community in plants from no apparent abiotic stress (NASP). However, previous studies report lower diversity in salt-stressed plants compared to unstressed plants (Hammami et al. 2016 ; Lu et al. 2022 ). Studies from additional plant species are necessary to establish if our results are specific to B. napus . Species accumulation curves do not reach stabilization, especially in plants from waterlogged and saline soils (Loro et al. 2012 ; Zhou et al. 2016 ; Santamaria et al. 2018 ). This suggests that the specific richness of these communities is even higher than the data presented here. Specifically, this richness appears to be underestimated in the endophytic communities of canola populations from saline soils. Canonical correspondence analysis (CCA) results indicate that the fungal endophytic leaf community of canola is not significantly correlated with environmental variables (field conditions). The confidence on the interpretation of this results could be limited by the fact that the endophytic leaf community of canola was rather scarce in soils with no apparent abiotic stress or that all the evaluated variables in the present study were related to soil, which does not seem to influence the composition of endophytic communities in leaves. When evaluating the soil-plant-foliar endophytes from an integrated approach, the data suggest that the fungal endophytic community in canola leaves could be modulated on the plant's response to abiotic stress mediated by soil (saline or waterlogged) rather than a direct effect of soil on the leaf endophytic community. This may also be explained by the type of transmission of the evaluated endophytes, which come from horizontal transmission and are influenced by environmental factors such as air and rain that can transport inocula to plant leaves. Regarding the soils parameters analyzed, it was observed that there is 10 times more sodium (Na + ) in the saline soil compared to the no apparent abiotic stress soil, and the Na + /K + ratio is 1.5 times higher in the saline soil compared to the no apparent abiotic stress soil. This indicates some ionic imbalance in these "saline" soils, even though all 3 soils are below what is considered saline or saline-sodic (FAO). From a plants physiological perspective, this SS ion imbalance may force the plant to adjust its ion balance by means of changes in the allocation of metabolic resources and the expression of ion pumps and other mechanisms of ionic homeostasis, among a plethora of physiological responses (Tyerman, 2018). The analysis of several plant parameters suggest that the observed ionic imbalance in field condition with saline soils causes changes in dry weight, water content, and chlorophyll concentration in the leaves of these canola populations, typical indicators of a situation of stress. The results showed that leaves from canola populations in field conditions with waterlogged soil exhibit a significantly lower dry weight (estimated as whole leaf dry weight) compared to canola populations from no apparent abiotic stress in soils. Previous studies have recorded that canola seedlings and plants display delayed growth, late development, and significantly reduced dry weight (Xu et al. 2015 ; Liang et al. 2023 ). Ploschuk et al. ( 2023 ) reported that canola plants experienced substantial reductions in leaf area index and foliage dry weight following waterlogging. The results obtained in this study are consistent with previous research, as biomass (whole leaf dry weight) is significantly lower under abiotic stress conditions. one of the causes of what was observed may be that in waterlogged soil is likely due to reduced root respiration caused by soil hypoxia and limitations in gas exchange generated by waterlogging (Casierra-Posada & Peña-Olmos 2022 ; Guo 2020; Casierra-Posada & Cutler 2017). However, the results presented here reveal that leaf water content is significantly higher in canola populations in saline soil (SSP) when compared to canola population in no apparent abiotic stress soil (NASP) when dry weight is normalized to disk area. This might be related to leaf thickness (data not shown). The data presented in Fig. 4a, indicates that the leaf dry weight of the different populations where leaves of plants grown without abiotic stress have 91.6% and 89% higher dry weight than leaves of plants grown on saline and waterlogged soils, respectively. On the other hand, the analyses indicate that canola populations under salinity stress (SSP) harbor a higher abundance of leaf endophytes than plants in field condition with no apparent abiotic stress soil (NASP). Direct correlation between higher water content and leaf endophyte abundance has been previously reported by D'Jonsiles et al. (2020) where increased water activity and endophyte presence were positively associated using the biospeckle technique in leaves of Jatropha curcas L. ( Euphorbiaceae ). Additionally, it is possible to observe in the field (com pers. Gaiser, data not shown), that the number of plants from the non-abiotic stressed oilseed rape population (NASP) was much higher than the cover of the abiotic stressed oilseed rape populations under field conditions (SSP and WSP), and plants from canola populations in waterlogged soils have a higher leaf water content compared to canola populations with no apparent abiotic stress soils. These results could indicate that endophytes contribute to plant adaptation to abiotic stress by regulating or modifying physiological, biochemical, and metabolic activities in plants. Since our approach could only identify endophytes present in living individuals (i.e. those who survived the abiotic stress factors), further experimental approaches using aforementioned endophyte species were in order to shed light on this hypothesis and to elucidate the specific physiological effects on the crop. The data here analysed indicate that the survival of plant populations in soils with abiotic stress (saline or waterlogged) may be related to the associated leaf endophytic community. According to Hyakumachi's review (2013), mechanisms involved in promoting plant growth by PGPF (Plant Growth-Promoting Fungi) include: (i) production of gibberellin-like substances, indoleacetic acid (IAA) or abscisic acid, (ii) cellulose and starch degradation, (iii) supply of mineral nutrients, (iv) suppression of harmful compounds from soil microorganisms, and (v) production of volatile substances. In this study, the characterization of the leaf endophytic fungal community of different canola populations was performed as an initial approach to detect strains associated with plants that survived and grew under abiotic stress. Thus, in the future, identifying the possible mechanisms involved in promoting the growth of populations found in the field modulated by these interactions. The yield of many Brassica species, such as rapeseed/canola ( Brassica napus L.), mustard ( Brassica juncea L.), cabbage ( Brassica oleracea L.), and turnip ( Brassica rapa L.), is highly affected by salinity (Jan et al. 2016b ). It has been previously reported that saline stress inhibits the growth and development of canola (Bacarin et al. 2011 ). Akhter et al. ( 2023 ) revealed that Super and Punjab varieties of B. napus significantly reduced biomass (expressed as dry weights of aerial parts and roots) under 200 mM salt conditions, and leaf thickness was significantly lower compared to the control without salinity. Comparing previous studies with the results of the present study, it can be concluded that soil salinity delays normal growth, development, and other physiological functions of the plant due to excessive accumulation of Na + and Cl − ions and nutrient deficiency (Garthwaite et al. 2005 ; Jan et al. 2016a , b ). The results obtained in this study show significantly lower values in chlorophyll a and total chlorophyll ( a + b ) when comparing canola populations implanted in saline soil with canola populations with no apparent abiotic stress. Previous reports indicate that saline stress induces a reduction in growth and yield of canola crops, often associated with a decrease in their photosynthetic capacity (Athar & Ashraf 2009 ; Athar et al. 2014 ; Akhter et al. 2023 ). The application of NaCl also decreases total chlorophyll concentration in Zea mays L. (maize) (Turan et al. 2009 ) and Helianthus annuus L. (sunflower) (Santos 2004 ). These reports align with the results obtained in this study, and several previous studies attribute the reduction in photosynthesis to saline stress, where the activity of Photosystem II (PSII) is inhibited, chlorophyll pigments are destroyed due to the accumulation of toxic ions, and CO 2 fixation rates are reduced (Athar et al. 2014 ; Bacarin et al. 2011 ). Regarding chlorophyll in canola populations in field condition with waterlogged soil, there is a trend towards lower values in chlorophyll a and total chlorophyll, although this result is not significant compared to canola populations with no apparent abiotic stress soil. However, Casierra-Posada & Cutler (2017) reported a reduction in chlorophyll content in Brassica oleracea var. capitata subjected to waterlogging. Other studies also recorded a significant decrease in chlorophyll content in leaves of rapeseed plants growing under waterlogging stress (Men et al. 2020 ; Habibzadeh et al. 2012 ). These studies agree with the observed trend of decreased chlorophyll concentration in WS seen in this work. The knowledge of the mycobiota associated with canola leaves and the interaction with the plant represents a fundamental approach to develop bio-inputs of agronomic interest that promote growth and development of canola, especially those that favor a better plant response to abiotic stress, making it possible to obtain a higher yield in fields with these crops. This study is a first approach towards understanding the endophytic community in canola. Future studies will seek to analyze the endophyte-plant interaction under abiotic stress, since it seems to promote improvements in the development and growth of canola under abiotic stress conditions. Declarations Supplementary Information The online version contains supplementary material available at https:// Author contributions All authors contributed to the study conception and design as well as to material preparation, data collection, and discussions. Molecular lab work was performed by Rocio F. Gaiser and Vanesa E. Tossi. Phylogenetic analyses were performed by Rocio F. Gaiser. The statistical analyses in this study were performed by Rocio F. Gaiser, Axel J. Rizzo and Carolina A. Robles. Macroscopic and microscopic analyses were performed by Rocio F. Gaiser and Cecilia C. Carmaran. Rocio F. Gaiser, Vanesa E. Tossi and Cecilia Carmarán were involved in planning and in the collection of material. Cecilia C. Carmaran designed and supervised the project, and revised the manuscript. The manuscript was written by Rocio F. Gaiser. All authors commented on the manuscript as well as read and approved the final version. Funding This study was financially supported by the National Scientific and Technical Research Council (CONICET) [PICT 202000513 and PICT 202001147]. The authors acknowledge the agribusiness Hórreos del Sudeste S.A. for their collaboration in the development of this study by providing B. napus seeds. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6708525","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":463542912,"identity":"afa20ffe-3d45-4b86-879a-01b4decfea73","order_by":0,"name":"Rocio Florencia Gaiser","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYJCCAww2DAxsINYHIGZjJ0pLGkQL4wyQFmai7EmDUMw8YJKAYvP25ocHPiTUyvPxHz722ebXNnk+ZgbGDx9zcGuROXPM4OCMhOOGbRJpybNz+24btjEzMEvO3IZbi4REDsNh3h/HGNskeIyZc3tuMwK1sDHzEtLCk3DMvo3//Gdmy57b9sRqqUlsY8hhZmb4cTuRsBYesF8OJAP9YszY23A7uY2ZsRm/X9ibH3/4kFBnO7//8GOGH39u285vbz744SMeLVBwGEIxtoHJBoLqgaAOSv8hRvEoGAWjYBSMNAAAH19N80G6BXkAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4647-4984","institution":"INMIBO CONICET UBA: Instituto de Micología y Botánica","correspondingAuthor":true,"prefix":"","firstName":"Rocio","middleName":"Florencia","lastName":"Gaiser","suffix":""},{"id":463542913,"identity":"b12dc459-f76c-4fcf-a0fe-08de8d770fd4","order_by":1,"name":"Vanesa Eleonora Tossi","email":"","orcid":"","institution":"INMIBO CONICET UBA: Instituto de Micología y Botánica","correspondingAuthor":false,"prefix":"","firstName":"Vanesa","middleName":"Eleonora","lastName":"Tossi","suffix":""},{"id":463542914,"identity":"6b5a4530-2556-420f-8354-7b0d05841a7a","order_by":2,"name":"Axel Joel Rizzo","email":"","orcid":"","institution":"INMIBO CONICET UBA: Instituto de Micología y Botánica","correspondingAuthor":false,"prefix":"","firstName":"Axel","middleName":"Joel","lastName":"Rizzo","suffix":""},{"id":463542915,"identity":"c4c82e1f-bb80-4d7f-b0d1-d243f14a2eb6","order_by":3,"name":"Carolina Analía Robles","email":"","orcid":"","institution":"INMIBO CONICET UBA: Instituto de Micología y Botánica","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"Analía","lastName":"Robles","suffix":""},{"id":463542916,"identity":"e8dc4458-00c2-4c86-a9bb-942bf9712f72","order_by":4,"name":"Cecilia Cristina Carmarán","email":"","orcid":"https://orcid.org/0000-0002-5078-390X","institution":"INMIBO CONICET UBA: Instituto de Micología y Botánica","correspondingAuthor":false,"prefix":"","firstName":"Cecilia","middleName":"Cristina","lastName":"Carmarán","suffix":""}],"badges":[],"createdAt":"2025-05-20 13:59:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6708525/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6708525/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83823240,"identity":"97498f48-35e9-441f-a5e2-1566d85c236c","added_by":"auto","created_at":"2025-06-03 09:24:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":63030,"visible":true,"origin":"","legend":"\u003cp\u003eThe Venn diagram represent the number of taxa corresponding to the endophytes obtained from oilseed rape plants in the three field conditions of the oilseed rape populations: without abiotic stress canola populations (NASP), saline soil canola populations (SSP) and waterlogged soil canola populations (WSP).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6708525/v1/c9df763e14ae478c38eaa93c.png"},{"id":83823393,"identity":"270c6a20-1e18-4ed4-b801-43a49c5dfe5b","added_by":"auto","created_at":"2025-06-03 09:32:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":74195,"visible":true,"origin":"","legend":"\u003cp\u003eAccumulation curves of endophytic species for each field condition.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6708525/v1/911498fe58a474f72a1e44b1.png"},{"id":83823395,"identity":"512e4ee3-6d7d-42a0-ace6-fd946f43d54b","added_by":"auto","created_at":"2025-06-03 09:32:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":113715,"visible":true,"origin":"","legend":"\u003cp\u003eBiplot of canonical correspondence analysis (CCA1 and CCA2 axes) ordination of endophytic fungal taxa (n = 18) in canola leaves. Species: \u003cem\u003eAlternaria \u003c/em\u003esp.1, \u003cem\u003eAlternaria \u003c/em\u003esp.2, \u003cem\u003eAlternaria \u003c/em\u003esp.3, \u003cem\u003eAlternaria \u003c/em\u003esp.4, \u003cem\u003eStemphylium \u003c/em\u003esp.1, \u003cem\u003eStemphylium \u003c/em\u003esp.2, \u003cem\u003eStemphylium \u003c/em\u003esp.3, \u003cem\u003eStemphylium \u003c/em\u003eaff. \u003cem\u003eamaranthi\u003c/em\u003e, and other taxa (morphotypes 1 to 9). Environmental parameters: pH, EC, Nt, Cot, OM, SAR, Ca\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Na\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, Cl\u003csup\u003e-\u003c/sup\u003e, SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e, CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e, HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e. Field conditions: no abiotic stress soil (NAS), saline soil (SS), and waterlogged soil (WS).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6708525/v1/cb0669ae7c34473b927e0e8a.png"},{"id":83823243,"identity":"d97f8e82-8994-4966-ac7b-d427aa594c9c","added_by":"auto","created_at":"2025-06-03 09:24:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":122998,"visible":true,"origin":"","legend":"\u003cp\u003ePlant parameters of each canola population growing in different field conditions. a) Dry weight of whole leaves; b) Water content of whole leaves (% relative to fresh weight); c) Dry weight of leaf discs; d) Chlorophyll \u003cem\u003ea\u003c/em\u003econcentration (µg/mg of fresh weight of the disc); e) Chlorophyll \u003cem\u003eb\u003c/em\u003e concentration (µg/mg of fresh weight of the disc); f) Total chlorophyll concentration (\u003cem\u003ea\u003c/em\u003e + \u003cem\u003eb\u003c/em\u003e) (µg/mg of fresh weight of the disc). NASP = no apparent abiotic stress canola population; SSP = saline soil canola population; WSP = waterlogged soil canola population. Error bars represent the standard error. Different letters denote significant differences between field conditions, when compared to NASP (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6708525/v1/da5797fac400184ec8acfadb.png"},{"id":85135958,"identity":"ed786f72-8188-4fd9-b1cb-15e7d08ca5ae","added_by":"auto","created_at":"2025-06-22 07:38:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1182749,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6708525/v1/850b5372-5950-4cf5-98c9-9b15bc381245.pdf"}],"financialInterests":"","formattedTitle":"Effect of abiotic stress on the endophytic fungal community in Brassica napus L. (winter canola) leaves","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFungal endophytes are microorganisms that infect healthy tissues of all plant lineages without causing any symptoms of disease (Arnold et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Suryanarayanan \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Horizontally transmitted endophytes are very diverse and mostly belong to the Phylum \u003cem\u003eAscomycota\u003c/em\u003e (Arnold and Lutzoni \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Endophytes can have a significant impact on plant communities by conferring tolerance to biotic and abiotic stress, increasing biomass and reducing water consumption, or decreasing fitness by altering resource allocation (Dickinson et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kumar and Nautiyal \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Rodriguez et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Previous studies (Fadiji and Babalola \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Grabka et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) even suggest that endophytes may contribute to the establishment of plant species by enhancing their competitiveness.These reports agree that these organisms play a fundamental role in plant responses to various external disturbances, which are significant at the ecological level. Current knowledge about plant adaptation to environmental changes not only suggests that plants can adapt, but also that they can interact, modify and select specific microbial communities to mitigate the negative effect of different stressors (Rodriguez and Dur\u0026aacute;n \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This protective function appears not to be carried out by all microorganisms, but rather by some microbial groups (Rodriguez and Dur\u0026aacute;n \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Considering the increasing number of studies supporting the idea that plants selectively interact with their surrounding microbiome (Rodriguez and Dur\u0026aacute;n \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Suryanarayanan and Shaanker \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), it becomes evident that the study of endophytic communities is of utmost importance to build a comprehensive map of interactions and their effects for species of interest.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBrassica napus\u003c/em\u003e L. (also known as rape, rapeseed and canola) is one of the most important oilseed crops worldwide, since the oil from its seeds is used in food industry and for biodiesel production, being also widely used in crop rotation and forage (G\u0026oacute;mez et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Card et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, among others).\u003c/p\u003e \u003cp\u003eTraditional rapeseed was found harmful for the animals that graze on it, since it caused myocardial alterations due to the presence of erucic acid. For this reason, the oil was destined for industrial purposes. It was not until 1970 that Canada and Europe achieved varieties improved, with low content of erucic acid and glucosinolates in the seeds. These varieties are called \u0026ldquo;00\u0026rdquo; or \u0026ldquo;double zero\u0026rdquo; in Europe and CANOLA (CANadian Oil Low Acid) in Canada. The production of rapeseed in the world was recorded at 87.2\u0026nbsp;million tons in the year 2022, while the record of the yield of rapeseed in the world is 21,824 (100g/ha) (FAO, 2022). In 2022, the continent with the highest production and yield of rapeseed was Europe, with 29.7\u0026nbsp;million tons and 29,197 (100g/ha) respectively. The second continent with the highest production was Asia, with 29.1\u0026nbsp;million tons of production and 17758 (100g/ha) of yield, and in the third place was America, with a production of 21.4\u0026nbsp;million tons and a yield of 21339 (100g/ha) (FAO, 2022). Canada is the world's leading country producer of canola, with a production of 18.7\u0026nbsp;million tons and a yield of 21,747 (100g/ha) (FAO, 2022).\u003c/p\u003e \u003cp\u003eIn Argentina, these varieties (\u0026ldquo;00\u0026rdquo; or CANOLA) introduced from Europe and Australia are currently used (S\u0026aacute;nchez-Vallduv\u0026iacute; and Chamorro \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The incorporation of this crop into Argentinian production schemes presents numerous advantages considering the improvements in genetics and management over the last decade, which have raised the average crop yields in the country to 1,800 kg/ha, compared to the historical 1,400 kg/ha (S\u0026aacute;nchez-Vallduv\u0026iacute; and Chamorro \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Schwab \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). According to FAO data (2022), the yield of canola was 20,482 (100g/ha) and production was 52,915 tons in 2022 for Argentina. Winter canola is an important alternative in agriculture and the oil industry in Argentina, since it supplies oil in the summer season and promotes rotation with other crops such as winter cereals (wheat, barley, oats) and does not compete in the oil industry with soybeans and sunflowers (Kirkegaard et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite these advantages, canola production has not reached a stable volume due to technological limitations regarding the improvement and management of the crop (de Emilio \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Iriarte and L\u0026oacute;pez \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). One of the factors that negatively affects canola crop is the abiotic salinity stress. The yield of many brassica species, such as canola (\u003cem\u003eBrassica napus\u003c/em\u003e), mustard (\u003cem\u003eBrassica juncea\u003c/em\u003e L.), cabbage (\u003cem\u003eBrassica oleracea\u003c/em\u003e L.) and turnip (\u003cem\u003eBrassica rapa\u003c/em\u003e L.), are highly affected by salinity (Jan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e). It has been previously reported that salt stress causes inhibition of canola growth and development (Akhter et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Bacarin et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe aim of this work is to characterize the fungal endophytic community of canola leaves (var. Hornet) grown under different field conditions (saline soil and waterlogged soil). This work also explores the relationships between plant parameters, soil parameters and endophytic diversity.\u003c/p\u003e \u003cp\u003eThe characterization of foliar endophytic communities associated with a crop such as canola is of great interest, promoting new technological approaches to crop stress tolerance through biologically active compounds and complexes and the study of their physiological effects. To our knowledge, the study by Zhang et al. (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) is the only one that studies the endophytic community of \u003cem\u003eB. napus\u003c/em\u003e grown in fields in China (leaves, stems and roots).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSite and soil characterization\u003c/h2\u003e \u003cp\u003eThe study was carried out in three canola populations located in a 35 ha. field in General Juan Madariaga, Province of Buenos Aires, Argentina (37\u0026deg; 01' 41.9\" S 57\u0026deg; 07' 56.7\" W). The soil was differentiated by three field conditions: no apparent abiotic stress (NAS), a saline condition according to local considerations and a frequently waterlogged condition (referred to as SS and WS, respectively). In each of these soil conditions different populations of canola developed and were subsequently analyzed (referred to as NASP, SSP and WSP, respectively).\u003c/p\u003e \u003cp\u003eSamples were taken from different zones under different conditions (NAS, SS and WS). Composite samples of each soil from 3 sampled zones were placed in bags. The 3 soils were characterized through analytical tests conducted at the Soil, Water, and Plant Laboratory, Soil Science Department, Faculty of Agronomy, University of Buenos Aires.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSampling and plant material\u003c/h3\u003e\n\u003cp\u003eThe studies were carried out on 3 populations of canola, variety Hornet (Al High Tech), planted in each of the previously described soils. The plants were sown simultaneously on April 13th, 2019, by the agribusiness H\u0026oacute;rreos del Sudeste S.A. Sampling was conducted 140 days after planting. Within each population (according to field conditions), plants were randomly selected, and fully expanded healthy young leaves were chosen. Four leaves per plant were sampled from 40 plants of each population, resulting in a total of 480 leaves selected from 120 healthy plants (without visible alterations or damage to the aerial part).\u003c/p\u003e\n\u003ch3\u003ePlant parameters\u003c/h3\u003e\n\u003cp\u003eTo calculate biomass, the largest leaf of the four leaves per plant was used. Biomass in canola leaves was estimated as the dry weight of whole leaves and as the dry weight of 7 mm diameter discs for each population.\u003c/p\u003e \u003cp\u003eFor water content, the same leaves used to calculate biomass were utilized. The water content of the canola leaves was calculated as a percentage relative to the fresh weight of the leaves:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:WC\\:=\\:\\frac{FW\\:-\\:DW}{FW}\\:*\\:100\\%$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere: WC\u0026thinsp;=\u0026thinsp;water content; FW\u0026thinsp;=\u0026thinsp;fresh weight; DW\u0026thinsp;=\u0026thinsp;dry weight.\u003c/p\u003e \u003cp\u003eA precision balance (Ohaus Traveler TA302, China) was used to obtain whole leaf fresh and dry weight and calculate their water content.\u003c/p\u003e \u003cp\u003eThe chlorophyll content was measured in one of the four sampled leaves per plant. Chlorophyll \u003cem\u003ea\u003c/em\u003e (Chl \u003cem\u003ea\u003c/em\u003e) and chlorophyll \u003cem\u003eb\u003c/em\u003e (Chl \u003cem\u003eb\u003c/em\u003e) were measured by immersing 7 mm diameter leaf discs in 1 mL of N-N dimethylformamide (DMF) solution. The discs were previously weighed on a precision balance (Ohaus Explorer, USA). They were then incubated in the dark at 4\u0026deg;C, and after 7 days, the absorbance of the solution was measured at 663.8 nm and 646.8 nm using a spectrophotometer (Shimadzu UV-1800, Kyoto, Japan) with quartz cuvettes. The concentration of chlorophyll was calculated in \u0026micro;g/mL according to the following equations (Porra 1989, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2002\u003c/span\u003e):\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\left[Chl\\:a\\right]\\:=\\:12{{A}^{663.8}\\:-\\:3.11A}^{646.8}$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:\\left[Chl\\:b\\right]\\:=\\:20.78{{A}^{646.8}\\:-\\:4.88A}^{663.8}$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\:[Chl\\:a+b]\\:=\\:17.67{{A}^{646.8}\\:+\\:7.12A}^{663.8}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe chlorophyll concentration was normalized to the DMF volume and to the weight of the discs (\u0026micro;g/mg of tissue, with tissue as the fresh weight of 7 mm diameter discs).\u003c/p\u003e\n\u003ch3\u003eIsolation and morphological identification of endophytic fungi\u003c/h3\u003e\n\u003cp\u003eThe leaves of the 120 selected plants were processed as follows: 1 leaf per plant was used to isolate fungal endophytes, recovering strains between 24 and 86 hours after sowing, to characterize the fungal communities. The leaf fragment, was superficially sterilized with 70% ethanol for 1 min, 4% Sodium Hypochlorite for 2 min, 50% ethanol 30 s and 3 washes with sterile distilled water for 2 min each, then the leaf fragment was left to dry on sterile paper before sowing it in Petri dishes. Isolates were obtained from 14\u0026ndash;15 fragments per leaf of 5 mm diameter in Petri dishes of 9 cm-diameter with 2% Malt Extract Agar with chloramphenicol (100 mg L\u0026thinsp;\u0026minus;\u0026thinsp;1) (2% MEAc) cultured at 23\u0026ndash;25\u0026deg;C in the dark.\u003c/p\u003e \u003cp\u003eAll strains were studied under an optical microscope (Olympus BX41, Tokyo, Japan) with an attached Infinity1 camera. The macromorphological characteristics of the colonies were also evaluated: color and texture of the mycelium, reverse of the culture, reproductive structures, edge of growth, among others. Taking these characteristics into consideration, the strains were grouped in morphotypes.\u003c/p\u003e\n\u003ch3\u003eDNA extraction, PCR and sequencing\u003c/h3\u003e\n\u003cp\u003eTwenty-nine representative strains of the most abundant morphotypes (morphotypes representing 95% of the obtained strains) were selected for DNA sequencing studies. These selected strains were cultured on 2% MEA and the cultures were incubated at 23\u0026ndash;25\u0026deg;C for 10\u0026ndash;21 days in the dark. Genomic DNA was extracted from fresh mycelial culture using the DNeasy UltraClean microbial kit (Qiagen, Germany), following the manufacturer's protocol. The ITS (Internal Transcribed Spacer) of the rRNA gene was amplified using the universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGTCTATTGATATGC-3') under the following PCR conditions: initial denaturation at 94\u0026deg;C for 3 min, 45 cycles of denaturation at 94\u0026deg;C for 45 s, annealing at 55\u0026deg;C for 45 s, extension at 72\u0026deg;C for 1 min, and a final extension at 72\u0026deg;C for 10 min. The PCR reactions were carried out in a final volume of 50 \u0026micro;L, containing 5 \u0026micro;L of 10x buffer, 0.2 mM of each dNTP, 0.4 \u0026micro;M of each primer, 0.5 \u0026micro;L of Taq DNA polymerase (Qiagen, Germany), 5 \u0026micro;L of fungal genomic DNA, and 36.5 \u0026micro;L of milliQ water. The RPB2 region (second largest subunit of RNA polymerase) was amplified using the primers RPB2\u0026ndash;5F2 (5'-GGGGWGAYCAGAAGAAGGC-3') and RPB2\u0026ndash;7cR (5'-CCCATRGCTTGTYYRCCCAT-3') under the following PCR conditions: 5 cycles of 45 s at 94\u0026deg;C, 45 s at 60\u0026deg;C, and 2 min at 72\u0026deg;C, followed by 5 cycles with a hybridization temperature of 58\u0026deg;C, followed by 30 cycles with a hybridization temperature of 54\u0026deg;C, and a final extension at 72\u0026deg;C for 7 min. The PCR reactions were carried out in a final volume of 50 \u0026micro;L, containing 5 \u0026micro;L of 10x buffer, 3 \u0026micro;L of Mg, 0.2 mM of each dNTP, 0.4 \u0026micro;M of each primer, 0.5 \u0026micro;L of Taq DNA polymerase (INBIO Highway, Argentina), 2 \u0026micro;L of fungal genomic DNA, and 36.5 \u0026micro;L of milliQ water. All PCR products were purified using the DNA PuriPrep-GP kit (INBIO HighWay, Argentina). All DNA fragments were sequenced by Macrogen Inc sequencing service (South Korea).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analyses\u003c/h2\u003e \u003cp\u003eThe obtained sequences were initially compared with sequences from the NCBI database (GenBank) using BLAST (Basic Local Alignment Search Tools) to search for highly similar sequences as the first step for subsequent phylogenetic studies (Supplementary material S1 to S12). The names of the species to which the reference sequences belong were maintained as they appear in GenBank.\u003c/p\u003e \u003cp\u003eThe DNA sequences were edited and manipulated using the BioEdit Sequence Alignment Editor program (Hall, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) and aligned with the MEGA-X program (Kumar et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) using the CLUSTAL option, leaving all parameters at their default values. Maximum parsimony (MP) trees were constructed using the MEGA-X program, with 1000 bootstrap replicates, and gaps were treated with 95% partial deletion. The evolutionary model selection and maximum likelihood (ML) phylogenetic analyses were conducted in IQ-TREE v. 2.2.0 (Minh et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The most appropriate model, according to Bayesian Information Criteria (BIC), was selected by the ModelFinder algorithm (Kalyaanamoorthy et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) included in IQ-TREE. Support for internal nodes was estimated using 1000 ultrafast bootstrap replicates (UFboot; Hoang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and the Shimodaira-Hasegawa approximate likelihood ratio test (SH-aLRT) with 1000 replicates. The command line used for the pre-aligned ITS1 and RPB2-5f matrices was: iqtree2 -s inputfile.nex -bb 1000 -alrt 1000 \u0026ndash;nt AUTO. According to BIC, the best-fitting substitution model selected for \u003cem\u003eAlternaria\u003c/em\u003e ITS was SYM\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G4 (i.e., symmetrical model, a proportion of invariable sites [I\u0026thinsp;=\u0026thinsp;0.532], and a Gamma distribution with four categories [alpha parameter\u0026thinsp;=\u0026thinsp;0.5]); the best-fitting substitution model selected for \u003cem\u003eStemphylium\u003c/em\u003e ITS was TIM2e\u0026thinsp;+\u0026thinsp;G4 (i.e., transition model, equal base frequency, and a Gamma distribution with four categories [alpha parameter\u0026thinsp;=\u0026thinsp;0.275]), and the best-fitting substitution model selected for \u003cem\u003eAlternaria\u003c/em\u003e RPB2 was TIM2e\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G4 (i.e., transition model, equal base frequency, a proportion of invariable sites [I\u0026thinsp;=\u0026thinsp;0.513], and a Gamma distribution with four categories [alpha parameter\u0026thinsp;=\u0026thinsp;1.05]). The trees were visualized using FigTree v.1.4.4 (Rambaut 2018), with branch lengths measured in the number of substitutions per site. Bayesian inference was conducted using MrBayes v. 3.2.7a program (Ronquist et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), with 5,000,000 generations, a chain temperature value set at 0.05, and a burn-in set at 25%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eSpecies accumulation curves and estimates of total fungal endophyte richness were inferred using EstimateS version 9.1.0 (Colwell \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Endophyte diversity was measured by the Shannon diversity index (H) using the same software. The similarity between the endophyte communities studied was evaluated with the S\u0026oslash;rensen similarity index using the following formula:\u003cdiv id=\"Eque\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Eque\" name=\"EquationSource\"\u003e\n$$\\:S\\:=\\frac{2C}{A\\:+\\:B}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere S is the degree of similarity, A and B are the number of endophytic species from two of the field conditions (NAS, SS, or WS) and C is the number of endophytic species common to both field conditions.\u003c/p\u003e \u003cp\u003eTo assess the influence of environmental variables (different field conditions) on the presence and frequency of endophytes (biodiversity of the foliar fungal endophyte community), a canonical correspondence analysis (CCA) was performed using R Core Team (2020). Hellinger transformation was conducted to correct potential statistical errors associated with rare species (Legendre \u0026amp; Gallagher \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStatistical analyses were performed in R version 4.0.3 (R Core Team, 2020). A one-factor ANOVA was conducted (alpha\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The plant parameters included leaf biomass, leaf disc biomass, leaf water content, chlorophyll \u003cem\u003ea\u003c/em\u003e, chlorophyll \u003cem\u003eb\u003c/em\u003e, and total chlorophyll (chlorophyll \u003cem\u003ea\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eb\u003c/em\u003e). The factor analyzed was field conditions (NAS, SS, and WS). Variance was modeled using a Generalized Least Squares (GLS) linear model, and maximum likelihood estimations were performed in cases where the assumption of homoscedasticity was not met (leaf biomass, leaf disc water content). The best model was chosen based on the Akaike Information Criterion (AIC). Pairwise comparisons were conducted using Tukey's Honestly Significant Difference (HSD) test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIdentification of endophytic fungi\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, 1787 leaf fragments were evaluated, obtaining a total of 231 strains, belonging to 3 genera and 8 species. A total of 21 strains were obtained from NASP leaves, 87 strains from SSP leaves, and 123 strains from WSP leaves. The genera \u003cem\u003eStemphylium\u003c/em\u003e Wallr. (\u003cem\u003ePleosporaceae, Ascomycota\u003c/em\u003e) and\u003cem\u003e\u0026nbsp;Alternaria\u003c/em\u003e Nees. (\u003cem\u003ePleosporaceae, Ascomycota\u003c/em\u003e) were the most abundantly represented, accounting for 95% of the strains (Table 1). In addition, Fig. 1 shows the distribution of taxa in each field condition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Number, relative frequencies and sample type of isolated strains of the three field conditions of canola populations: without abiotic stress canola populations (NASP), saline soil canola populations (SSP) and waterlogged soil canola populations (WSP). Other taxa correspond to a total of 9 morphotypes. In the first column Genbank accession numbers of each strain are given between parentheses.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"600\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 354px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTaxon (strain GenBank accession number)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 166px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of strains\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRelative frequency\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNASP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSSP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWSP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 354px;\"\u003e\n \u003cp\u003e\u003cem\u003eAlternaria\u003c/em\u003e sp.1\u003c/p\u003e\n \u003cp\u003e(ITS/RPB2: PP949836/-, PP949839/-, PP949840/PP960212, PP949842/PP960214)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e7.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 354px;\"\u003e\n \u003cp\u003e\u003cem\u003eAlternaria\u003c/em\u003e sp.2\u003c/p\u003e\n \u003cp\u003e(ITS: PP949838/PP960211, -/PP960217)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e6.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 354px;\"\u003e\n \u003cp\u003e\u003cem\u003eAlternaria\u0026nbsp;\u003c/em\u003esp.3\u003c/p\u003e\n \u003cp\u003e(ITS/RPB2: PP949841/PP960213, PP949843/PP960215, PP949844/PP960216)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 354px;\"\u003e\n \u003cp\u003e\u003cem\u003eAlternaria\u0026nbsp;\u003c/em\u003esp.4\u003c/p\u003e\n \u003cp\u003e(ITS/RPB2: PP949837/PP960210)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 354px;\"\u003e\n \u003cp\u003e\u003cem\u003eStemphylium\u003c/em\u003e sp.1\u003c/p\u003e\n \u003cp\u003e(PP950360, PP950361, PP950364, PP950365, PP950370, PP950377, PP950378).\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e60.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 354px;\"\u003e\n \u003cp\u003e\u003cem\u003eStemphylium\u0026nbsp;\u003c/em\u003esp.2\u003c/p\u003e\n \u003cp\u003ePP950366, PP950367, PP950368, PP950371, PP950373, PP950375, PP950376)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e10.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 354px;\"\u003e\n \u003cp\u003e\u003cem\u003eStemphylium\u003c/em\u003e sp.3\u003c/p\u003e\n \u003cp\u003e(PP950369, PP950372)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 354px;\"\u003e\n \u003cp\u003e\u003cem\u003eStemphylium\u0026nbsp;\u003c/em\u003eaff.\u003cem\u003e\u0026nbsp;amaranthi\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e(PP950362, PP950363, \u0026nbsp;PP950374)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 354px;\"\u003e\n \u003cp\u003eOther taxa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e5.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 354px;\"\u003e\n \u003cp\u003eTOTAL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e123\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e102\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eIn order to improve the taxonomic identifications, a total of 3 matrices with 442 sequences were analyzed (68 sequences for the ITS gene from strains assigned to \u003cem\u003eStemphylium\u0026nbsp;\u003c/em\u003espp., 186 sequences for the ITS gene and 188 sequences for the RPB2 gene from strains assigned to \u003cem\u003eAlternaria\u0026nbsp;\u003c/em\u003espp.), including the 37 sequences obtained from the 29 strains obtained in this study. Characterization using molecular markers and phylogenetic analyses placed the strains recovered during this study within the genera \u003cem\u003eAlternaria\u0026nbsp;\u003c/em\u003eand \u003cem\u003eStemphylium\u003c/em\u003e. Phylogenetic analyses (Supplementary material S1 to S6) grouped the \u003cem\u003eAlternaria\u0026nbsp;\u003c/em\u003estrains within the section \u003cem\u003eAlternaria\u003c/em\u003e, distinguishing the sequences obtained into 4 different species, without a clear grouping to a particular species within the section.\u003c/p\u003e\n\u003cp\u003eThe phylogenetic trees corresponding to the genus \u003cem\u003eAlternaria\u003c/em\u003e with the ITS gene and analysis reveal that the section \u003cem\u003eAlternaria\u003c/em\u003e is a sister clade of the group formed by the section \u003cem\u003eAlternantherae\u003c/em\u003e and the section \u003cem\u003eSonchi\u003c/em\u003e under maximum parsimony (bootstrap support of 93%) (see Supplementary material S6); under maximum likelihood, the section \u003cem\u003eAlternaria\u003c/em\u003e is positioned as a sister clade of the section \u003cem\u003eSonchi\u003c/em\u003e but without robust support values (see Supplementary material S5); under Bayesian inference, the section \u003cem\u003eAlternaria\u003c/em\u003e is a sister clade of the section \u003cem\u003eSonchi\u003c/em\u003e, but without posterior probability values exceeding the minimum value to consider that clade as true under the study conditions (see Supplementary material S4). With this marker, species-level identification was not achieved for any of the strains in this study.\u003c/p\u003e\n\u003cp\u003eWhen analyzing the phylogenetic trees obtained for the genus \u003cem\u003eAlternaria\u003c/em\u003e with the RPB2 gene, it is observed that the section \u003cem\u003eAlternaria\u003c/em\u003e is positioned as a sister clade of the section \u003cem\u003eAlternantherae\u003c/em\u003e under maximum parsimony (bootstrap of 98%), maximum likelihood (SH-aLRT = 99.9% and UFB = 100%), and under Bayesian inference (posterior probability of 100%) (see Supplementary material S1, S2, and S3).\u003c/p\u003e\n\u003cp\u003eThe phylogenetic trees obtained for the \u003cem\u003eStemphylium\u0026nbsp;\u003c/em\u003egenus were inconclusive, as the supports and posterior probabilities are low. Additionally, all 3 trees exhibit polytomies. However, it can be observed that strains 3, 7, and 25 group together with \u003cem\u003eStemphylium amaranthi\u003c/em\u003e Y.F. Pei \u0026amp; X.G. Zhang in all 3 topologies, and strain 2 is positioned within the clade of \u003cem\u003eStemphylium vesicarium\u003c/em\u003e (Wallr.) E.G. Simmons with a support of 98% under maximum parsimony (see Supplementary material S7, S8, and S9). For these results, three strains were identified as belonging to \u003cem\u003eStemphylium\u003c/em\u003e aff. \u003cem\u003eamaranthi\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiversity and species composition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe canola populations from soils with abiotic stress (SSP and WSP) exhibited the highest species richness among the studied populations, with a total of 14 species in the canola SSP and 11 species in the WSP. Meanwhile, the canola population from no apparent abiotic stress (NASP) presented 3 endophytic species. The highest estimated species richness (Bootstrap Mean) was found in the canola population from saline soil (17.2), followed by the waterlogged soil canola population (12.6), and the without abiotic stress canola population (3.4). The Shannon-Wiener index (H) gave 0.67 for NASP, 1.51 for WSP and 1.52 for SSP. Similarity between oilseed rape populations under different field conditions (S\u0026oslash;rensen\u0026apos;s index), gave 0.42 between NASP and SSP, 0.5 between NASP and WSP and 0.67 between SSP and WSP. The species accumulation curves of the canola populations growing in different field conditions are shown in Fig. 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of environmental factors and endophytic fungal diversity\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoil characterization\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe analyses of the three soils belonging to the area where canola populations were sampled (WAS, SS and WS) are shown in Supplementary material S13.\u003c/p\u003e\n\u003cp\u003eThe canonical correspondence analysis (CCA) was conducted to detect the variance explained by environmental variables (field conditions) in the structure of the endophytic fungal community in canola leaves in terms of the presence and frequency of endophytes. The ordination plot was generated to visualize the associations of fungal communities with environmental predictors (Fig. 3). In Fig. 3, the first and second axes explained 65% and 35% of the variance in the biodiversity of the foliar fungal endophytic community, respectively. The factors pH and EC (soil electrical conductivity) contributed most to the differences between the foliar fungal endophytic communities and the environmental factors. The first axis (CCA1) positively correlated with EC, and the second axis (CCA2) positively correlated with soil pH. Although the CCA was not statistically significant (p \u0026gt; 0.05), a trend can be observed indicating that there is higher richness of endophytic species on canola leaves under abiotic stress field conditions (saline and waterlogged).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAbiotic stresses such as waterlogging and salinity negatively impact plant growth and development, affecting chlorophyll levels, as well as the production and quality of agricultural products. In this work, different plant parameters were analyzed using healthy leaf samples from different populations of canola: a) biomass in leaf (expressed as dry weight in grams); b) biomass in leaf discs (expressed as dry weight in milligrams); c) water content in whole leaves (expressed as a percentage relative to fresh weight); d) concentration of chlorophyll \u003cem\u003ea\u003c/em\u003e; e) concentration of chlorophyll \u003cem\u003eb\u003c/em\u003e; f) concentration of chlorophyll \u003cem\u003ea\u0026nbsp;\u003c/em\u003e+ \u003cem\u003eb\u003c/em\u003e (total). Fig. 4 showed the obtained data from each population.\u003c/p\u003e\n\u003cp\u003eFig. 4a shows that the weight of dry leaves from NASP is significantly higher than that of the leaves from the canola populations growing under abiotic stress (SSP and WSP) and the dry weight of whole leaves of SSP being significantly lower than WSP and NASP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe water content of whole leaves of SSP and WSP is significantly higher than the water content of leaves from NASP (Fig. 4b).\u003c/p\u003e\n\u003cp\u003eDry weight of leaf disks from canola populations under saline stress is significantly higher than dry weight of leaf disks from canola populations growing under waterlogging stress and without abiotic stress (Fig. 4c). There are no significant differences between the dry weight of leaf disks from canola populations in waterlogged soil and those from canola populations without abiotic stress.\u003c/p\u003e\n\u003cp\u003eAs observed in Fig. 4d and f, the concentration of chlorophyll\u003cem\u003e\u0026nbsp;a\u003c/em\u003e and total chlorophyll (\u003cem\u003ea\u003c/em\u003e + \u003cem\u003eb\u003c/em\u003e) is lower in the leaves of canola populations from saline and waterlogged soils compared to leaves from plants grown without abiotic stress.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this work, we studied the fungal endophytic community colonizing leaves of \u003cem\u003eB. napus\u003c/em\u003e, from three populations subjected to different field conditions, by performing analyses on soil and plant parameters and their associated endophytes.\u003c/p\u003e \u003cp\u003eThe results showed the presence of two genera: \u003cem\u003eAlternaria\u003c/em\u003e and \u003cem\u003eStemphylium\u003c/em\u003e. The molecular and phylogenetic analyses using molecular markers improve the identification of strains belonging to these genera. Regarding the genus \u003cem\u003eAlternaria\u003c/em\u003e, phylogenetic topology placed the strains from this study within the section \u003cem\u003eAlternaria\u003c/em\u003e, and the phylogenetic hypothesis suggests the presence of more than one species belonging to \u003cem\u003eAlternaria\u003c/em\u003e. The section \u003cem\u003eAlternaria\u003c/em\u003e appears as the sister clade of the section \u003cem\u003eAlternantherae\u003c/em\u003e. This result is in agreement with the results reported by Lawrence et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e and Woudenberg et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, and \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2015\u003c/span\u003e. The phylogenetic trees obtained for the genus \u003cem\u003eStemphylium\u003c/em\u003e were inconclusive, as the supports and posterior probabilities are low. Additionally, all analyzed trees exhibit polytomies. However, it can be observed that strains 3, 7, and 25 cluster together with \u003cem\u003eStemphylium amaranthi\u003c/em\u003e in all topologies and therefore could be assigned to \u003cem\u003eStemphylium\u003c/em\u003e aff. \u003cem\u003eamaranthi\u003c/em\u003e. Within the genus \u003cem\u003eStemphylium\u003c/em\u003e, 28 species can be distinguished based on regions of the ITS, GAPDH, and cmdA genes (Woudenberg et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). More genes and further studies are needed to improve the taxonomic placement of obtained strains.\u003c/p\u003e \u003cp\u003eThis study shows that 20.1% of the fungal strains in total canola leaves (NASP) and 12.6% of the fungal strains on the leaves of the canola population under salinity stress (SSP) belong to the genus \u003cem\u003eAlternaria\u003c/em\u003e section \u003cem\u003eAlternaria.\u003c/em\u003e Previously this genus had been registered as endophytes of \u003cem\u003eB. napus\u003c/em\u003e (Zhang et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Khalmuratova et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) studied the endophytic community of roots of halophytic plants, \u003cem\u003eSuaeda australis\u003c/em\u003e Moq., \u003cem\u003eSuaeda maritime\u003c/em\u003e (L.) Dumort., and \u003cem\u003ePhragmites australis\u003c/em\u003e (Cav.) Trin. ex Steud., and obtained 42%, 53%, and 33% root isolates with endophytic colonization by \u003cem\u003eAlternaria alternata\u003c/em\u003e (Fr.) Keissl., this being the most abundant endophyte in these plants. The results presented here appear to associate the presence of the genus \u003cem\u003eAlternaria\u003c/em\u003e as predominant under abiotic stress conditions (salinity and waterlogging) and higher endophytic diversity than under no apparent abiotic stress conditions. The high diversity may be due to environmental conditions (salinity or waterlogging) playing a determining role in leaf susceptibility to endophytic organisms, as abiotic stress may be a necessary factor for establishment in the host plant. Thus, \u003cem\u003eAlternaria\u003c/em\u003e seems to be a frequent endophyte associated with plants grown in saline stress environments (Bouzouina et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Khalmuratova et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study shows that the genus \u003cem\u003eStemphylium\u003c/em\u003e is highly represented in the leaves of the three canola populations studied, including the canola population growing under salinity. \u003cem\u003eStemphylium\u003c/em\u003e contains 96 species and includes plant pathogenic, endophytic, and saprophytic fungi with worldwide distributions. Unlike \u003cem\u003eAlternaria\u003c/em\u003e there are a few reports of this genus as endophyte, however have been isolated as endophytes from marine and terrestrial habitat. \u003cem\u003eStemphylium solani\u003c/em\u003e G.F. Weber was isolated as a fungal endophyte of the leaves from the medicinal plant \u003cem\u003eArtemisia absinthium\u003c/em\u003e L. and the bioactive ethyl acetate fungal extract showed antifungal, insect antifeedant and nematicidal activity (Diaz et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Goncalves et al. (2021) shows that inoculation of the salt marsh plant \u003cem\u003eSalicornia\u003c/em\u003e sp. with a root isolate of the genus \u003cem\u003eStemphylium\u003c/em\u003e positively influenced total biomass production and nitrogen concentration in roots under salinity condition that was optimal for plant growth (150 mM NaCl). The data obtained in our work, in terms of the representation of \u003cem\u003eStemphylium\u003c/em\u003e in the populations studied, could suggest that strains belonging to this genus could be responsible for the plant responses registered in saline field conditions.\u003c/p\u003e \u003cp\u003eOne of the most interesting results obtained is the diversity (expressed as the Shannon index) index value which reaches 1.52 in saline soils, which is much more diverse than the endophytic community in plants from no apparent abiotic stress (NASP). However, previous studies report lower diversity in salt-stressed plants compared to unstressed plants (Hammami et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Lu et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Studies from additional plant species are necessary to establish if our results are specific to \u003cem\u003eB. napus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eSpecies accumulation curves do not reach stabilization, especially in plants from waterlogged and saline soils (Loro et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Santamaria et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This suggests that the specific richness of these communities is even higher than the data presented here. Specifically, this richness appears to be underestimated in the endophytic communities of canola populations from saline soils.\u003c/p\u003e \u003cp\u003eCanonical correspondence analysis (CCA) results indicate that the fungal endophytic leaf community of canola is not significantly correlated with environmental variables (field conditions). The confidence on the interpretation of this results could be limited by the fact that the endophytic leaf community of canola was rather scarce in soils with no apparent abiotic stress or that all the evaluated variables in the present study were related to soil, which does not seem to influence the composition of endophytic communities in leaves. When evaluating the soil-plant-foliar endophytes from an integrated approach, the data suggest that the fungal endophytic community in canola leaves could be modulated on the plant's response to abiotic stress mediated by soil (saline or waterlogged) rather than a direct effect of soil on the leaf endophytic community. This may also be explained by the type of transmission of the evaluated endophytes, which come from horizontal transmission and are influenced by environmental factors such as air and rain that can transport inocula to plant leaves.\u003c/p\u003e \u003cp\u003eRegarding the soils parameters analyzed, it was observed that there is 10 times more sodium (Na\u003csup\u003e+\u003c/sup\u003e) in the saline soil compared to the no apparent abiotic stress soil, and the Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e ratio is 1.5 times higher in the saline soil compared to the no apparent abiotic stress soil. This indicates some ionic imbalance in these \"saline\" soils, even though all 3 soils are below what is considered saline or saline-sodic (FAO). From a plants physiological perspective, this SS ion imbalance may force the plant to adjust its ion balance by means of changes in the allocation of metabolic resources and the expression of ion pumps and other mechanisms of ionic homeostasis, among a plethora of physiological responses (Tyerman, 2018).\u003c/p\u003e \u003cp\u003eThe analysis of several plant parameters suggest that the observed ionic imbalance in field condition with saline soils causes changes in dry weight, water content, and chlorophyll concentration in the leaves of these canola populations, typical indicators of a situation of stress. The results showed that leaves from canola populations in field conditions with waterlogged soil exhibit a significantly lower dry weight (estimated as whole leaf dry weight) compared to canola populations from no apparent abiotic stress in soils. Previous studies have recorded that canola seedlings and plants display delayed growth, late development, and significantly reduced dry weight (Xu et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Liang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Ploschuk et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) reported that canola plants experienced substantial reductions in leaf area index and foliage dry weight following waterlogging. The results obtained in this study are consistent with previous research, as biomass (whole leaf dry weight) is significantly lower under abiotic stress conditions. one of the causes of what was observed may be that in waterlogged soil is likely due to reduced root respiration caused by soil hypoxia and limitations in gas exchange generated by waterlogging (Casierra-Posada \u0026amp; Pe\u0026ntilde;a-Olmos \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Guo 2020; Casierra-Posada \u0026amp; Cutler 2017). However, the results presented here reveal that leaf water content is significantly higher in canola populations in saline soil (SSP) when compared to canola population in no apparent abiotic stress soil (NASP) when dry weight is normalized to disk area. This might be related to leaf thickness (data not shown). The data presented in Fig.\u0026nbsp;4a, indicates that the leaf dry weight of the different populations where leaves of plants grown without abiotic stress have 91.6% and 89% higher dry weight than leaves of plants grown on saline and waterlogged soils, respectively. On the other hand, the analyses indicate that canola populations under salinity stress (SSP) harbor a higher abundance of leaf endophytes than plants in field condition with no apparent abiotic stress soil (NASP). Direct correlation between higher water content and leaf endophyte abundance has been previously reported by D'Jonsiles et al. (2020) where increased water activity and endophyte presence were positively associated using the biospeckle technique in leaves of \u003cem\u003eJatropha curcas\u003c/em\u003e L. (\u003cem\u003eEuphorbiaceae\u003c/em\u003e). Additionally, it is possible to observe in the field (com pers. Gaiser, data not shown), that the number of plants from the non-abiotic stressed oilseed rape population (NASP) was much higher than the cover of the abiotic stressed oilseed rape populations under field conditions (SSP and WSP), and plants from canola populations in waterlogged soils have a higher leaf water content compared to canola populations with no apparent abiotic stress soils. These results could indicate that endophytes contribute to plant adaptation to abiotic stress by regulating or modifying physiological, biochemical, and metabolic activities in plants. Since our approach could only identify endophytes present in living individuals (i.e. those who survived the abiotic stress factors), further experimental approaches using aforementioned endophyte species were in order to shed light on this hypothesis and to elucidate the specific physiological effects on the crop.\u003c/p\u003e \u003cp\u003eThe data here analysed indicate that the survival of plant populations in soils with abiotic stress (saline or waterlogged) may be related to the associated leaf endophytic community. According to Hyakumachi's review (2013), mechanisms involved in promoting plant growth by PGPF (Plant Growth-Promoting Fungi) include: (i) production of gibberellin-like substances, indoleacetic acid (IAA) or abscisic acid, (ii) cellulose and starch degradation, (iii) supply of mineral nutrients, (iv) suppression of harmful compounds from soil microorganisms, and (v) production of volatile substances. In this study, the characterization of the leaf endophytic fungal community of different canola populations was performed as an initial approach to detect strains associated with plants that survived and grew under abiotic stress. Thus, in the future, identifying the possible mechanisms involved in promoting the growth of populations found in the field modulated by these interactions.\u003c/p\u003e \u003cp\u003eThe yield of many \u003cem\u003eBrassica\u003c/em\u003e species, such as rapeseed/canola (\u003cem\u003eBrassica napus\u003c/em\u003e L.), mustard (\u003cem\u003eBrassica juncea\u003c/em\u003e L.), cabbage (\u003cem\u003eBrassica oleracea\u003c/em\u003e L.), and turnip (\u003cem\u003eBrassica rapa\u003c/em\u003e L.), is highly affected by salinity (Jan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e). It has been previously reported that saline stress inhibits the growth and development of canola (Bacarin et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Akhter et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) revealed that Super and Punjab varieties of \u003cem\u003eB. napus\u003c/em\u003e significantly reduced biomass (expressed as dry weights of aerial parts and roots) under 200 mM salt conditions, and leaf thickness was significantly lower compared to the control without salinity. Comparing previous studies with the results of the present study, it can be concluded that soil salinity delays normal growth, development, and other physiological functions of the plant due to excessive accumulation of Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e ions and nutrient deficiency (Garthwaite et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Jan et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003eb\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe results obtained in this study show significantly lower values in chlorophyll \u003cem\u003ea\u003c/em\u003e and total chlorophyll (\u003cem\u003ea\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eb\u003c/em\u003e) when comparing canola populations implanted in saline soil with canola populations with no apparent abiotic stress. Previous reports indicate that saline stress induces a reduction in growth and yield of canola crops, often associated with a decrease in their photosynthetic capacity (Athar \u0026amp; Ashraf \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Athar et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Akhter et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The application of NaCl also decreases total chlorophyll concentration in \u003cem\u003eZea mays\u003c/em\u003e L. (maize) (Turan et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and \u003cem\u003eHelianthus annuus\u003c/em\u003e L. (sunflower) (Santos \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). These reports align with the results obtained in this study, and several previous studies attribute the reduction in photosynthesis to saline stress, where the activity of Photosystem II (PSII) is inhibited, chlorophyll pigments are destroyed due to the accumulation of toxic ions, and CO\u003csub\u003e2\u003c/sub\u003e fixation rates are reduced (Athar et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Bacarin et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegarding chlorophyll in canola populations in field condition with waterlogged soil, there is a trend towards lower values in chlorophyll \u003cem\u003ea\u003c/em\u003e and total chlorophyll, although this result is not significant compared to canola populations with no apparent abiotic stress soil. However, Casierra-Posada \u0026amp; Cutler (2017) reported a reduction in chlorophyll content in \u003cem\u003eBrassica oleracea\u003c/em\u003e var. \u003cem\u003ecapitata\u003c/em\u003e subjected to waterlogging. Other studies also recorded a significant decrease in chlorophyll content in leaves of rapeseed plants growing under waterlogging stress (Men et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Habibzadeh et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These studies agree with the observed trend of decreased chlorophyll concentration in WS seen in this work.\u003c/p\u003e \u003cp\u003eThe knowledge of the mycobiota associated with canola leaves and the interaction with the plant represents a fundamental approach to develop bio-inputs of agronomic interest that promote growth and development of canola, especially those that favor a better plant response to abiotic stress, making it possible to obtain a higher yield in fields with these crops. This study is a first approach towards understanding the endophytic community in canola. Future studies will seek to analyze the endophyte-plant interaction under abiotic stress, since it seems to promote improvements in the development and growth of canola under abiotic stress conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e The online version contains supplementary material available at https://\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e All authors contributed to the study conception and design as well as to material preparation, data collection, and discussions. Molecular lab work was performed by Rocio F. Gaiser and Vanesa E. Tossi. Phylogenetic analyses were performed by Rocio F. Gaiser. The statistical analyses in this study were performed by Rocio F. Gaiser, Axel J. Rizzo and Carolina A. Robles. Macroscopic and microscopic analyses were performed by Rocio F. Gaiser and Cecilia C. Carmaran. Rocio F. Gaiser, Vanesa E. Tossi and Cecilia Carmar\u0026aacute;n were involved in planning and in the collection of material. Cecilia C. Carmaran designed and supervised the project, and revised the manuscript. The manuscript was written by Rocio F. Gaiser. All authors commented on the manuscript as well as read and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This study was financially supported by the National Scientific and Technical Research Council (CONICET) [PICT 202000513 and PICT 202001147]. The authors acknowledge the agribusiness H\u0026oacute;rreos del Sudeste S.A. for their collaboration in the development of this study by providing \u003cem\u003eB. napus\u003c/em\u003e seeds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e The datasets generated during the current study are available in the Genbank repository.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e This article does not contain any studies with animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAkhter N, Noreen A, Saifullah S, Noman A, Shahnaz MM, Letuma PM, Kausar A, Siddique M, Hashem M, Alamri S, Al-zoubi OM, Saleem M, Khalid N, Aqeel M (2023) Salt ion mediated changes in biochemical and anatomical characteristics of \u003cem\u003eBrassica napus\u003c/em\u003e can be countered with Moringa Leaf extract. 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Stud Mycol 75:171\u0026ndash;212. https://doi.org/10.3114/sim0015 \u003c/li\u003e\n\u003cli\u003eXu M, Ma H, Zeng L, Cheng Y, Lu G, Xu J, Zhang X, Zou X (2015) The effect of waterlogging on yield and seed quality at the early flowering stage in \u003cem\u003eBrassica napus\u003c/em\u003e L. Field Crops Research 180:238\u0026ndash;245. https://doi.org/10.1016/j.fcr.2015.06.007 \u003c/li\u003e\n\u003cli\u003eZhang Q, Zhang J, Yang L, Zhang L, Jiang D, Chen W, Li G (2014) Diversity and biocontrol potential of endophytic fungi in \u003cem\u003eBrassica\u003c/em\u003e \u003cem\u003enapus\u003c/em\u003e. Biol Control 72:98\u0026ndash;108. https://doi.org/10.1016/j.biocontrol.2014.02.018 \u003c/li\u003e\n\u003cli\u003eZhou S, Qiu H, Feng C, Guo Y, Wang X, Chen C (2016) Impact of deltamethrin on the endophytic fungal community of a Chinese cabbage, \u003cem\u003eBrassica chinensis\u003c/em\u003e. Chem Ecol 32(3):259\u0026ndash;269. https://doi.org/10.1080/02757540.2015.1135907 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Winter canola, Alternaria, Stemphylium, abiotic stress, fungal endophytes","lastPublishedDoi":"10.21203/rs.3.rs-6708525/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6708525/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWinter canola is an oleaginous plant whose seeds are used in the food and biodiesel industries. Fungal endophytes are found in asymptomatic tissues of almost all plant lineages, promoting positive responses to stress situations. This study aims to characterize the fungal endophytic community of canola leaves (var. Hornet) grown under abiotic stress, comparing them with those plants with no apparent stress signs. Our results showed that canola has a community of endophytic fungi distributed in the Phylum Ascomycota, with members of \u003cem\u003eAlternaria\u003c/em\u003e and \u003cem\u003eStemphylium\u003c/em\u003e as dominant groups. We found 231 endophytic fungi strains, belonging to 2 genera and 29 fungal morphotypes. A greater diversity and richness of endophytic fungi were found in canola populations grown on saline soil (14 species, SSP) and waterlogged soil (11 species, WSP). Conversely, the canola population with no apparent abiotic stress (NASP) had only 3 endophytic species. The highest estimated species richness was found in the SSP (17.2), followed by the WSP (12.6), and the NASP (3.4). We also observed NASP showed very low endophyte colonization compared to stressed plants, being 6 and 4 times higher on WSP and SSP, respectively. Additionally, plant parameter analyses showed that canola leaves of SSP and WSP accumulated more water than canola leaves of NASP. The results obtained contribute to the knowledge on the dynamics of the endophytic fungal community associated with canola cultivation in the field, allow estimation of mycodiversity, and might be useful in research aimed at crop growth promotion.\u003c/p\u003e","manuscriptTitle":"Effect of abiotic stress on the endophytic fungal community in Brassica napus L. (winter canola) leaves","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-03 09:24:40","doi":"10.21203/rs.3.rs-6708525/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6d47045f-aff3-402b-afc4-31e3df38bd3c","owner":[],"postedDate":"June 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-22T07:30:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-03 09:24:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6708525","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6708525","identity":"rs-6708525","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}