Inuence of genome methylation of g tree accessions on the natural nematode and rust incidence

Brazil is the largest g producer in South America, but the Brazilian commercial g tree cultivation is based on the planting of a single cultivar, ‘Roxo-de-Valinhos’, resulting in serious problems related to diseases. Since there are epigenetic variations in the plant-pathogen interaction, mainly through gene regulation, the aim of this study was to carry out the in vivo characterization of g accessions through the analysis of the natural root-knot nematode and leaf rust incidence correlated to its epigenomic prole, in order to support conservation works and genetic improvement. Regarding the analysis of the presence of nematodes, it was observed that all plants were attacked by this pathogen, and the identication of Meloidogyne incognita as the root-knot nematode species was conrmed. However, the rust incidence and the global genomic methylation content where statistical different between evaluated accessions. The joint analysis of data showed that methylation and the leaf rust incidence, when observed in the same phenological phase of plants, are correlated, presenting evidences of the same factorial pressure loads in genotypes, with the premise of similar behavior in these genotypes. Biotic factors are also responsible for changes in the DNA methylation of plants, demonstrating a positive role in promoting plant defense.


Introduction
Common g tree, Ficus carica L, commonly known as g, is a deciduous fruit tree species belonging to the Moraceae family, whose center of origin is the Mediterranean region (Patil 2020), but widely cultivated in all regions of the world with subtropical/temperate climate (Usai et al. 2020).
With important global economic expression, being marketed mainly in the fresh fruit and dried fruit market (Gurung et al. 2021), g is among the twenty main fresh fruits exported by Brazil, which stands out as the main producer of the southern hemisphere, with production of approximately 23 thousand tons per year in 2,208 hectares of planted area. However, the Brazilian commercial g tree cultivation is based on the planting of a single cultivar, 'Roxode-Valinhos', resulting in serious problems related to pests and diseases, which reduce production and depreciate fruits, reducing pro ts, impairing plant development and demanding high expenses with inputs and cultural treatments ).
Among the main diseases affecting g crops, g tree rust, caused by the fungus Cerotelium ci (Solano-Baéz et al. 2017) and root-knot nematodes (Meloidogyne incognita) (Abdel-Sattar et al. 2020) stand out, imposing a major challenge in most cultivated areas around the world.
Thus, studies aimed at nding solutions to some of these problems would bring an enormous contribution to the development of the g culture, with genotypic diversi cation being an important strategy for the crop, and genetic diversity being an important tool for the survival of the species and adaptation to biotic and abiotic exposures (Dooren et al. 2020), the latter being characterized by the phenotypic plasticity of plants.
In addition, the strong selection pressures imposed by extreme environments and specialized niches can sometimes lead to the independent evolution of similar morphological and/or physiological characteristics in different lineages (Sackton and Clark 2019), being organisms adapted to extreme environments especially interesting to study the fundamental questions of evolutionary biology.
Given the above, the aim of this study was to carry out the in vivo characterization of g accessions through the analysis of the natural root-knot nematode and leaf rust incidence correlated to its epigenomic pro le, in order to support conservation works, genetic improvement and crop production

Results And Discussion
In evaluations carried out in relation to the root-knot nematode incidence, it could be observed that all plants were attacked by this pathogen, showing their susceptibility to it. Analysis regarding the presence of nematodes identi ed 120-150 adult nematodes per root sample and 15 Meloidogyne sp adults per soil sample and 60 Helicotylenchus sp. adults per root and soil samples.
The identi cation of root-knot nematode species (Meloidogyne incognita) was con rmed under photonic microscope, by examining and documenting the perineal pattern (females) and the morphology of the labial region of males, as shown in Figure 1. Other Ficus species were selected regarding their resistance to these nematodes to compensate for the current lack of resistant germplasm in the cultivated g tree. Despite the high resistance and graft compatibility in accessions of Ficus racemosa tropical species (Cohn and Duncan 1990), no effective control strategy has yet been implemented, showing that the control of M. incognita is a key point for g tree cultivation (Neugebauer et al. 2018).
Thus, the control of cultivation environments (soil improvement and sterilization by sunlight), the treatment of plant diseases using chemical nematicides and biological control of root-knot nematodes using microorganisms (Du et al. 2020;Sikandar et al. 2020) or plant extracts are prophylactic measures that should be adopted for g cultivation (Tariq et al. 2017).
Regarding the rust incidence, Table 2 shows the leaf area percentages covered with Cerotelium ci pustules, where statistical difference could be observed between evaluated accessions, with emphasis on  "Roxo-de-Valinhos", considered the base cultivar for the comparison of crop quality in Brazil, presented between 3.9 and 27.3% of rust lesions, corroborating results found by Mezzalira et al. (2015), who compared the e ciency of alternative fungicides and insecticides in relation to conventional products registered for g tree culture in the state of Paraná and veri ed the incidence of the disease in this cultivar with approximate values ranging from 4.39 to 30% of incidence for control treatment.
In observed signi cant difference between plants and also between locations, concluding that accessions less susceptible to the disease may be associated with greater plant rusticity, but also that the diversity between them can generate genotype-environment interaction and that environmental conditions may be more favorable to the development of g trees naturalized in this environment in which genotypes had lower incidence.
The ability of a single genotype to generate alternative phenotypes based on changes in the environment -or phenotypic plasticity -is a potential mechanism by which plants In this sense, DNA methylation can be considered the best characterized epigenetic mechanism, which is  Accessions whose absolute values are above the upper limit line, represented by accessions 5, 6, 7, 10, 12 revealing that, in this case, irradiation was an external factor capable of changing these patterns in these plants, including in the DNA demethylation process. Figure 3 presents the principal component analysis using variables percentages of rust incidence in g accessions and their global genomic methylation content, demonstrating that methylation and rust incidence in young leaves are correlated, with the premise of similar behavior in genotypes.
As for the rust incidence in medium and basal leaves, no correlation with the methylation content was observed, which can be explained by the fact that the DNA of accessions was extracted from new leaves, indicating that, before leaf development, there is correlation positive between methylation and this disease; however, as the leaf matures, the global genomic methylation content possibly changes, making it impossible to correlate the variables observed at different phenological times. Thus, it appears that differences were observed in the rust incidence and in the global methylation content of the DNAs of the different g tree accessions belonging to the Active Germplasm Bank evaluated in this study. In addition, it was possible to correlate the disease with methylation, when observed in the same phenological phase of the plant, showing initial evidence of same factorial pressure loads in genotypes, indicating that, in addition to the genetic factor, Biotic factors are also responsible for changes in the DNA methylation of plants, demonstrating a positive role in promoting plant defense.
Thus, it is evident that future studies on the gene expression between treatments is an extremely important strategy for the understanding of complex regulatory systems, leading to the identi cation of genes of agronomic interest for the g tree crop, enabling its subsequent manipulation and propagation of improved cultivars for commercial purposes.

Methods
The plant material was composed of 44 Ficus sp. accessions belonging to the three-year-old Active Germplasm Bank of the Agricultural Sector, Faculty of Agrarian and Technological Sciences FCAT / UNESP, Campus of Dracena-SP.
The experimental design was randomized blocks, consisting of 44 treatments, each accession being considered a treatment, with two blocks and three plants per plot, totaling 270 plants, grown in 1.5 mx 1.5 m spacing, conducted in an open cup format, with 6 productive branches each. Treatments evaluated, with their respective accessions and places of origin, are presented in Table 1. Evaluations to quantify the natural rust incidence in the eld were carried out in November, coinciding with the beginning of fruit maturation. The experimental design adopted was completely randomized, each material being considered a treatment, with six replicates, with each plant being considered a replicate. Evaluation of the resistance of g accessions was carried out based on the infection e ciency, which was evaluated through the percentage of area with symptoms, taking into account a 1cm 2 disk of a speci c leaf region (upper right part of the leaf), in three leaves per replicate, each leaf being collected from one part of the plant (new, medium and basal leaves). The injured area was estimated with a 20 x magnifying glass coupled to a digital camera, and then, images were evaluated in computer based on methodology described by Rodrigues et al. (2012).
To quantify the global genomic methylation content of g trees, young leaves without spots or perforations were collected and washed under running water, and their veins were removed. The extraction of total genomic DNA from plant tissues was performed according to the modi ed cetyltrimethylammonium bromide technique.
Global DNA methylation was quanti ed using the Imprint DNA Methylation Quanti cation kit® (Sigma), following manufacturer's instructions. This kit functions similarly to an ELISA plate assay, using well strips that have been pre-treated with binding methylated DNA, and using DNA methylation sensitive capture antibody and detection allowing colorimetrically detection of relative amounts of DNA methylation at absorbance of 450nm.
Statistical analyses were carried out on results of all accessions in each evaluation data. The averages obtained were compared using Scott-Knott's test at 5% probability signi cance threshold. The SISVAR software (Ferreira 2019) was used to perform statistical analyses and experimental planning.
Subsequently, with data on variables percentage of rust incidence and global genomic methylation content, principal component analysis was performed to de ne the clustering pattern between genotypes and the relative contribution of diversity per character in order to allow identifying which variables overlap and that can be discarded through the residual correlation matrix obtained by the joint analysis of   Principal component analysis using percentages of rust incidence and global genomic methylation content of g tree accessions.