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 identified 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 identification of root-knot nematode species (Meloidogyne incognita) was confirmed 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.
Ferreira et al. (2015) evaluated the hostability to the Meloidogyne incognita nematode in fig tree accessions belonging to the Germplasm Bank of the Seedling Production Center of the Integral Technical Assistance Coordination (CATI) in São Bento do Sapucaí, state of São Paulo and found that diversity did not influenced the hostability of plants to the nematode, since the population of M. incognita eggs and juveniles was statistically equal in all accessions evaluated, and the total nematode population ranged from 40,000 to 105,000 per gram of extracted root.
Costa et al. (2015) evaluated 6 fig tree genotypes, including those evaluated in the present study, regarding the response to Meloidogyne javanica, M. incognita and M. enterolobii nematodes, with the possibility of being used as rootstock resistant to this pathogen, and concluded that all genotypes under study behaved as susceptible to them.
Rodrigues et al. (2012) selected mutants in fig plants formed by cuttings irradiated with gamma rays in order to increase their genetic variability in relation to vegetative and reproductive development and found that all plants were carriers of Meloidogyne incognita nematode, showing that they are susceptible to it, and it is not possible to select plants resistant to this disease, corroborating data of the present work.
In general, a variety of methods have been used to limit Meloidogyne damage, including the development of resistant cultivars (Park et al. 2020). Thus, the identification and characterization of natural sources of resistance are important steps for the development of control strategies for root-knot nematodes. In woody crops, genetically resistant rootstocks different from the cultivar of agronomic interest can be obtained (Saucet et al. 2016); however, in the specific case of fig trees, this strategy is not yet viable.
Other Ficus species were selected regarding their resistance to these nematodes to compensate for the current lack of resistant germplasm in the cultivated fig 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 fig 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 fig cultivation (Tariq et al. 2017).
Regarding the rust incidence, Table 2 shows the leaf area percentages covered with Cerotelium fici pustules, where statistical difference could be observed between evaluated accessions, with emphasis on Calimyrna, Genovesco, Roxo-de-Valinhos A, Stanford, White Adriatic, White Genova A, White Genova B, Smyrna B, Brunswich, PI 214, Accession 33, Accession 35, Accession 46, Caprifigo B, Accession 41, Accession 42, Mini Figo and Turco accessions, which obtained the lower rust incidence averages, indicating good adaptability to the climate conditions of the subtropical cwa climate type (mild and dry winters followed by very hot summers), according to the Köeppen classification (1948).
Table 2
Percentages of area covered with Cerotelium fici pustules in new (FFN), median (FFM) and basal (FFB) leaves of fig tree accessions.
TREATMENT | ACCESSION | FFN (%) | FFM (%) | FFB (%) | Mean |
1 | Calimyrna | 0 a | 13 a | 18 a | 10.6 a |
2 | Nobile | 1 a | 27 b | 25 b | 17.4 a |
3 | Genovesco | 0 a | 6 a | 20 a | 8.70 a |
4 | Roxo-de-Valinhos A | 0 a | 0 a | 12 a | 3.9 a |
5 | Stanford | 0 a | 1 a | 5 a | 1.90 a |
6 | White Adriatic | 3 a | 19 a | 20 a | 14.1 a |
7 | Bonato | 6 a | 23 a | 87 c | 38.7 b |
8 | White Genova A | 0 a | 8 a | 14 a | 7.40 a |
9 | White Genova B | 0 a | 1 a | 23 a | 8.20 a |
10 | Smyrna A | 13 a | 34 b | 77 c | 41.3 b |
11 | Smyrna B | 2 a | 5 a | 14 a | 6.80 a |
12 | Brunswich | 1 a | 0 a | 7 a | 2.60 a |
13 | Caprifig A | 20 a | 27 b | 45 b | 30.6 b |
14 | Pingo de Mel | 27 b | 0 a | 3 a | 10.0 a |
15 | Roxo-de-Valinhos Gigante | 43 b | 3 a | 58 b | 34.8 b |
16 | Palestino | 2 a | 14 a | 44 b | 20.0 a |
17 | Troyano | 5 a | 14 a | 32 b | 16.9 a |
18 | Vermelho | 2 a | 39 b | 70 c | 36.8 b |
19 | Irradiated Plant 440 | 0 a | 65 c | 40 b | 35.0 b |
21 | Irradiated Plant 189 | 60 c | 5 a | 90 c | 51.7 b |
22 | Irradiated Plant 214 | 20 a | 5 a | 15 a | 13.3 a |
23 | Irradiated Plant 301 | 0 a | 25 b | 90 c | 38.3 b |
24 | Nazaret | 0 a | 40 b | 60 c | 33.3 b |
25 | Cuello Negro | 0 a | 50 b | 0 a | 16.7 a |
26 | Roxo-de-Valinhos B | 7 a | 41 b | 23 a | 23.7 a |
27 | Accession 27 | 0 a | 15 a | 48 b | 21.1 a |
28 | Accession 28 | 0 a | 20 a | 80 c | 33.3 a |
29 | Accession 29 | 5 a | 8 a | 33 b | 15.6 a |
30 | Accession 30 | 2 a | 43 b | 43 b | 29.4 b |
31 | Accession 31 | 0 a | 0 a | 35 b | 11.7 a |
32 | Accession 32 | 0 a | 28 b | 70 c | 32.5 b |
33 | Accession 33 | 12 a | 8 a | 20 a | 13.30 a |
34 | Accession 34 | 2 a | 3 a | 37 b | 13.90 a |
35 | Accession 35 | 0 a | 3 a | 5 a | 2.50 a |
36 | Accession 36 | 2 a | 15 a | 45 b | 20.6 a |
37 | Accession 47 | 10 a | 2 a | 43 b | 18.3 a |
38 | Accession 44 | 0 a | 3 a | 43 b | 15.6 a |
39 | Accession 46 | 0 a | 3 a | 5 a | 2.50 a |
40 | Caprifig B | 0 a | 7 a | 8 a | 5.00 a |
41 | Accession 41 | 2 a | 0 a | 0 a | 0.60 a |
42 | Accession 42 | 0 a | 5 a | 5 a | 3.30a |
43 | Mini Figo | 8 a | 23 a | 13 a | 15.0 a |
44 | Preto | 3 a | 37 b | 35 b | 25.0 b |
45 | Turco | 0 a | 0 a | 8 a | 2.50 a |
*Different letters in the column differ statistically from each other by the Scott-Knott test at 0.05% probability. |
“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 efficiency of alternative fungicides and insecticides in relation to conventional products registered for fig tree culture in the state of Paraná and verified the incidence of the disease in this cultivar with approximate values ranging from 4.39 to 30% of incidence for control treatment.
In general, the mean values of the experiment ranged from 0.6 to 51.7%, corresponding to Accession 41 and Irradiated Plant (PI) 189 accessions, respectively. However, even the highest mean value is below the highest degrees of disease severity observed in literature. Sol-Rodríguez et al. (2021) evaluated the disease incidence and severity in fig trees in Mexico under the moisture conditions similar to those of the present experiment and observed C. fici incidence values between 66.2 and 96.2%.
Pastore et al. (2015) evaluated the resistance of fig tree accessions to rust in different locations and observed significant 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 fig 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 can respond quickly to external changes (Arnold et al. 2019), in which growth responses to competition and defense responses to the attack of organisms are two classic examples of adaptive phenotypic plasticity of plants (Fernández-Milmanda et al. 2020).
In this sense, DNA methylation can be considered the best characterized epigenetic mechanism, which is involved in many important aspects of the evolutionary biology of the fig tree, such as in the varietal and behavioral differentiation of plants under environmental pressures (Rodrigues et al. 2019).
DNA methylation is a conserved epigenetic marker that regulates several processes, such as gene silencing, genome stability and genomic imprinting (Zhang et al. 2018). It is also present in gene coding regions in many plant species, leading to their overexpression (Choi et al. 2020).
Figure 2 shows the quantification of global genomic methylation of evaluated fig tree accessions, where dashed horizontal lines show values that were statistically different from the main commercial fig cultivar in Brazil, 'Roxo-de-Valinhos', represented by accession number 4, whose absolute value was 0.172.
Accessions whose absolute values are above the upper limit line, represented by accessions 5, 6, 7, 10, 12, 13, 14, 15, 18, 19, 20, 34, 39 and 40, namely Stanford, Adriático Branco, Bonato, Smyrna, Brunswich, Caprifigo IAC, Pingo de Mel, Roxo-de-Valinhos Gigante, Figo Vermelho, PI 440, PI 189, Accession 35 from Monte Alto, Caprifigo ISA and Accession 41 from Monte Alto, respectively, present global genomic methylation content statistically higher than that of ‘Roxo-de-Valinhos’, indicating that, in relation to the selected parameter, they are hypomethylated.
The absolute values observed below the lower limit line, represented by accessions 22 and 26, respectively ‘PI 433’ and accession 27 from Monte Alto, present global genomic methylation content statistically lower than that of ‘Roxo-de-Valinhos’, indicating that, in relation to the selected parameter, they are hypermethylated, corroborating results found by Rodrigues et al., 2015, who, evaluating the same irradiated plants analyzed in the present work, also observed that accessions numbers 440 and 189 had higher methylation content when compared to ‘Roxo-de-Valinhos’, and accession 433 had lower content, 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 fig 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.
Several studies have established the role of epigenetic variations in the plant-microbe interaction, mainly through gene regulation (Kumar and Mohapatra 2021). Epigenetic mechanisms associated with interactions between plants and pathogens, in particular bacterial and fungal pathogens, demonstrate the positive role they can play in promoting plant defense (Zhu et al. 2015); however, the role of such change in DNA methylation in preparing plants against pests/diseases is not yet known (Wang et al. 2019).
Evidence shows that stress alters the epigenetic profile of plants, which can improve their stress tolerance capacity (Varotto et al. 2020), which may be DNA hyper- or hypomethylation, varying among species.
DNA hypomethylation is reported as a general feature in the promoter of many genes associated with fruit ripening, as they contain binding sites for transcription factors associated with ripening (LANG et al. 2017). However, Huanghuan et al. (2019) analyzed the influence of global DNA methylation on the ripening process of orange fruits and observed that DNA hypermethylation is critical for proper fruit ripening.
Thus, it appears that differences were observed in the rust incidence and in the global methylation content of the DNAs of the different fig 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 identification of genes of agronomic interest for the fig tree crop, enabling its subsequent manipulation and propagation of improved cultivars for commercial purposes.