Differential gene expression in maize seeds in interactions with Fusarium verticillioides, Aspergillus flavus and Penicillium sp.ABSTRACT:
Maize seed quality can be compromised by pathogenic fungi, making it essential to understand seed-pathogen interactions for the development of faster and more precise detection techniques. This study aimed to evaluate the physiological and sanitary quality of maize seeds and to determine whether suppression subtractive hybridization (SSH) can identify differentially expressed genes in interactions with F. verticillioides, A. flavus, and Penicillium sp., with validation by qRT-PCR. Seeds of the single-cross hybrid BM 810 were inoculated for 48 and 96 hours using the water restriction method and subjected to germination, first germination count, fungal incidence, tetrazolium, and germination speed index (GSI) tests. Based on the physiological and sanitary results, the following treatments were selected for the construction of six subtractive libraries: control (T), 96 h without fungus (96), 96 h with F. verticillioides (96F), Penicillium sp. (96P), and A. flavus (96A). Seed physiological and sanitary quality was negatively affected by fungal infection. The SSH technique proved efficient in detecting differentially expressed genes, with anthranilate synthase, cysteine proteinase, catalase B, glyceraldehyde-3-phosphate dehydrogenase, and superoxide dismutase 4 standing out as potential markers associated with fungal infection in maize seeds.
Index terms:
qRT-PCR; seed health quality; suppression subtractive hybridization; Zea mays L
RESUMO:
A qualidade das sementes de milho pode ser comprometida por fungos patogênicos, tornando essencial a compreensão das interações semente-patógeno para o desenvolvimento de técnicas de detecção mais rápidas e precisas. Objetivou-se avaliar a qualidade fisiológica e sanitária de sementes de milho e verificar se a técnica de hibridização subtrativa supressiva (SSH) é capaz de identificar genes diferencialmente expressos nas interações com F. verticillioides, A. flavus e Penicillium sp., com validação por qRT-PCR. Sementes do híbrido simples BM 810 foram inoculadas por 48 e 96 horas pelo método de restrição hídrica e submetidas aos testes de germinação, primeira contagem, incidência de fungos, tetrazólio e índice de velocidade de germinação (IVG). Com base nos resultados fisiológicos e sanitários, selecionaram-se os tratamentos testemunha (T), 96 h sem fungo (96), 96 h com F. verticillioides (96F), Penicillium sp. (96P) e A. flavus (96A) para a construção das seis bibliotecas subtrativas. A qualidade fisiológica e sanitária das sementes foi comprometida pela infecção fúngica. A técnica SSH mostrou-se eficiente na detecção de genes diferencialmente expressos, destacando-se antranilato sintase, cisteína proteinase, catalase B, gliceraldeído-3-fosfato desidrogenase e superóxido dismutase 4 como potenciais marcadores associados à infecção fúngica em sementes de milho.
Termos para indexação:
qRT-PCR; qualidade sanitária de sementes; hibridização subtrativa supressiva; Zea mays L
INTRODUCTION
The seed is considered the raw material that transports all the genetic potential of the species to the field. For this, it needs to have high physical, physiological, genetic and sanitary quality, the last of which referring mainly to the absence of pathogens (Ramos et al., 2014; Catão et al., 2020).
In Brazil, among the various phytopathogenic fungi that compromise quality and are most associated with maize (Zea mays L.) seeds are Fusarium verticillioides (Sousa et al., 2022), Penicillium sp. (Zacharias et al., 2024) and Aspergillus flavus (Morena et al., 2025). In general, the association of these fungi compromises the physiological quality of seeds. F. verticillioides, as a field fungus, can also favor the dispersion of the pathogen over long distances and its transmission from seed to plant (Sousa et al., 2022). A. flavus and Penicillium spp., fungi typically associated with storage, act mainly in seed deterioration under inadequate temperature and humidity conditions and can also compromise germination (Otero et al., 2020; Morena et al., 2025). In addition, these pathogenic fungi in seeds and grains produce mycotoxins that are harmful to human and animal health due to the toxic activity they can exert on the body (Akhtar and Kumar, 2024).
For these reasons, the detection and diagnosis of these pathogens in seeds becomes increasingly important. The criteria used for the detection of fungi in seeds follow the rules approved by the Manual of Seed Health Analysis and, for the most part, consist of stimulating the microorganisms to produce typical structures formed on or around the seeds (Brasil, 2009). However, the development of fast and safe detection methodologies is increasingly important, as it makes it possible to speed up decision-making regarding the commercialization and disposal of seed lots. It is worth mentioning that chemical treatment with eradicating and/or protective agents should not be conditioned exclusively on the verification of transmission of the pathogen by the seed, since soil fungi can infect seeds and cause seed rot, pre- and post-emergence damping-off and root rot, especially under cold and humid soil conditions, justifying protection measures regardless of the route of transmission (Lamichhane et al., 2020).
In this context, the identification of specific genes expressed by the seed/pathogen interaction is important for understanding the mechanisms that govern this infection, enabling the development of methods for early detection of the presence of these microorganisms in seed lots, through the marking or identification of specific proteins of this interaction (Martín et al., 2022). To identify differentially expressed genes, one method used is Suppressive Subtractive Hybridization (SSH), followed by qRT-PCR. SSH aims to identify and enrich rare and differentially expressed mRNA fragments. Subtractive hybridization comes from a methodology directed at selecting only the singular species of cDNA, removing the common sequences that are expressed (Chandrasekaran et al., 2019).
SSH has already been used, for example, to identify and quantify differentially expressed genes associated with citrus decline (Abreu et al., 2015), formation of sclerotia of Morchella conica (Chen et al., 2014), and interactions of tomato with Trichoderma longibrachiatum (De Palma et al., 2016). Thus, the qRT-PCR technique combined with the suppressive subtractive hybridization method can be an alternative in studies of expression of proteins involved in the mechanism of infection of maize seeds by pathogens, which are related to the beginning of the deterioration process.
In view of the above, the aim of this study was to evaluate the physiological and sanitary quality of maize seeds and to verify the efficiency of the suppressive subtractive hybridization (SSH) technique in the detection of differentially expressed genes in the interactions with F. verticillioides, A. flavus and Penicillium sp., with validation by expression analysis via qRT-PCR.
MATERIAL AND METHODS
The fungi F. verticillioides, A. flavus and Penicillium sp. were isolated from maize seeds using the Blotter test technique (Brasil, 2009). After identifying the colonies, they were transferred to Petri dishes containing PDA (potato-dextrose-agar) medium and kept in a growth chamber at 25 °C for seven days. Pure cultures were preserved until the moment of their use. Two hundred hybrid BM 810 maize seeds, previously sterilized with a 1% sodium hypochlorite solution, washed and dried on filter paper, were used in the experiment (Figure 1). The experimental design was completely randomized (CRD), with five treatments for each fungus evaluated (F. verticillioides, A. flavus and Penicillium sp.) and four replications. The treatments were: control [seeds treated with fungicide Carbendazim+Thiram (600 mL.100 kg⁻¹ of seeds)], without exposure to the culture medium, with the objective of inhibiting fungal growth; seeds without inoculation, kept in culture medium under water restriction for 48 hours; seeds without inoculation, kept in culture medium under water restriction for 96 hours; seeds inoculated with the respective fungus in culture medium under water restriction for 48 hours; and seeds inoculated with the respective fungus in culture medium under water restriction for 96 hours.
Inoculation was carried out using the method of water restriction on agar substrate (Machado, 2000), with a water potential of -1.0 MPa, obtained by adding 2% mannitol to the PDA medium. The seeds remained in the culture media in the presence or absence of fungal mycelia for 48 and 96 hours. Immediately after the composition of the treatments, the seeds were placed to dry for 24 hours under aseptic laboratory conditions (Figure 1).
Experimental flowchart of maize seed infection by fungi and gene expression analysis by SSH and qRT-PCR.
The physiological and sanitary quality of the seeds was determined by germination, vigor and health tests (blotter test). Germination test was carried out using four replications of 50 seeds of each treatment, evenly distributed on two sheets of germination paper, moistened with distilled water in the proportion of 2.5 times the mass of the dry paper, and placed to germinate at 25 °C, with a photoperiod of 12 hours, with counting performed at four (first germination count) and seven (germination) days after sowing (Brasil, 2025). The results were expressed as a percentage. The germination speed index (GSI) was evaluated simultaneously with the germination test, and the number of germinated seedlings was computed daily, at the same time. The index was calculated with the formula proposed by Maguire (1962).
The tetrazolium test was performed with four replications of 25 seeds, packed in paper rolls moistened with water for 18 hours at 25 °C. Subsequently, the seeds were longitudinally sectioned and immersed in a 0.5% 2,3,5-triphenyl tetrazolium chloride solution for 6 hours at 30 °C, in the absence of light. After this period, the seeds were washed in running water and evaluated according to Brasil (2025), and the results were expressed as a percentage of viable seeds (França-Neto et al., 2020).
Seed health was evaluated by the Blotter test with freezing (Brasil, 2025), using eight replications of 25 seeds distributed in Petri dishes (15 cm in diameter) containing three sheets of filter paper sterilized and moistened with sterile distilled water. The seeds were incubated at 25 °C with a photoperiod of 12 hours for 24 hours, transferred to a freezer (-20 °C) for 24 hours and kept at 25 ± 2 °C for five days. After incubation, the seeds were examined under stereoscopic microscope to evaluate the incidence of F. verticillioides, A. flavus and Penicillium sp. with the results expressed as percentage.
The data were subjected to analysis of variance (ANOVA), and the means were compared by the Scott-Knott test at 5% significance level, with the aid of R software (R Core Team, 2025).
For the construction of the subtractive libraries (SSH), treatments with 96 hours of inoculation were selected based on the results of physiological and sanitary tests, which had a greater impact on seed quality in this period compared to 48 hours. Thus, the selected treatments were: seeds treated with fungicide (T); seeds without inoculation under water restriction for 96 hours (96); seeds inoculated under water restriction for 96 hours with F. verticillioides (96F); seeds inoculated under water restriction for 96 hours with Penicillium sp. (96P); and seeds inoculated under water restriction for 96 hours with A. flavus (96A). The (T) and (96) treatments were used as “drivers”, while the (96F), (96P) and (96A) treatments were “testers”. Each “driver” was tested with each of the “testers”, with library 1 (TF) = T-96F; library 2 (TP) = T-96P; library 3 (TA) = T-96A; library 4 (96F) = 96-96F; library 5 (96P) = 96-96P; and library 6 (96A) = 96-96A (Figure 1).
For RNA extraction, the samples were macerated in liquid nitrogen, and then the PureLink Plant RNA reagent (Invitrogen™) was added. Total RNA was treated with the Amplification Grade DNase I (Invitrogen™) for elimination of residual genomic DNA, and the mRNA was purified with the GenElute™ mRNA Miniprep kit (Sigma-Aldrich), following the manufacturers’ specifications. The six subtractive suppression libraries were built with the PCR-Select™ cDNA Subtraction kit (Clontech), from 2 μg of mRNA per sample, according to the manufacturer’s protocol. The cDNA was digested with the RsaI enzyme for generation of blunt-end fragments, and then adapters 1 and 2R were bound to the digested cDNA “tester”. Subtractive hybridizations and suppressive PCR were then performed, aiming at the enrichment of differentially expressed sequences. The subtracted cDNAs were cloned with the TOPO TA vector (Invitrogen™ TOPO TA Cloning Kit) and transformed into DH5α™ T1 Phage Resistant E. coli (Promega).
After cloning, the transformed cells were incubated for 12 hours in liquid LB medium (Sambrook et al., 1989), and the plasmids were extracted with QIAprep Spin Miniprep Kit (Qiagen). The cDNAS with the sequences of interest were sequenced by GENEWIZ Inc. (USA). The primers M13 Forward and Reverse were used, with analysis by means of the SEQ sequence and the ABI chromatogram. After sequencing, the contigs were assembled using the DNA Baser Sequence Assembler (http://www.dnabaser.com) program and the vector sequences extracted with VecScreen. The sequences obtained were identified by similarity using BLASTn in the databases of the National Center for Biotechnology Information (NCBI).
Gene expression in maize seeds
For the validation of gene expression by qRT-PCR, protein-coding genes identified by similarity in the maize genome database were selected from each library. Based on these sequences, the primers were designed using the Primer Express 3.0 software (Applied Biosystems) (Table 1).
Protein-coding genes selected from each library (B) for gene expression analysis in qRT-PCR.
The qRT-PCR reactions were conducted in the ABI PRISM 7500 Real-Time PCR system (Applied Biosystems), with detection by SYBR Green. The thermal program consisted of: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C, with a final stage of 15 s at 95 °C. At the end of the amplification, the specificity of the reactions was confirmed by the analysis of the denaturation curve (60-95 °C), which revealed the presence of a single amplification product. The data were collected and stored in the 7500 Fast Software program (version 2.1). Each reaction was composed of 1.0 μL of cDNA (1:5 dilution), 0.2 μL of each forward and reverse primer (10 μM), 5.0 μL of SYBR Green Master Mix (Applied Biosystems) and 3.6 μL of ultrapure water. Negative controls and melting curves were included in all analyses.
Three biological repeats and three technical repeats were performed for each gene evaluated. The results were normalized by the threshold cycle (Ct) of the ubiquitin (UBI) and alcohol dehydrogenase (ADH) reference genes. Previously, a validation experiment confirmed that the amplification efficiencies of the target genes and the reference genes were similar and close to 100%, which allows the use of the comparative method of relative quantification, and the optimal cDNA dilution (1:5) for each reaction was determined. The samples with the lowest expression level for each gene were used as a calibrator and the relative expression was calculated by the mathematical model proposed by Pfaffl (2001).
RESULTS AND DISCUSSION
No significant differences were found in the first germination count between the inoculated and control treatments for any of the fungi evaluated (Table 2). However, in the final germination, reductions in the percentage of normal seedlings were observed as a function of time and the inoculated fungus, indicating that the effects of pathogens on the physiological quality of the seeds are more evident throughout the germination process than in the initial phases evaluated by the first count. In the tetrazolium test there was no statistical difference in seed quality, except for maize seeds inoculated for 96 hours with Penicillium sp. (Table 2).
The efficiency of the inoculation process was confirmed by the results of the sanitary test (Table 2). In inoculated seeds, there was a high incidence of the respective fungi, with values of 90% and 100% for F. verticillioides after 48 and 96 hours, 69% and 78% for A. flavus, and 24% and 27% for Penicillium sp., respectively. In the control and in seeds without inoculation, the incidence was equal to zero, demonstrating that the water restriction method was efficient in promoting the infection of the seeds by the pathogens in the established periods and that the fungicide treatment applied to the control was effective in inhibiting fungal development.
After 96 hours of inoculation with F. verticillioides there was a lower percentage of normal seedlings, which allows us to infer that this fungus is harmful to the quality of seed lots from a certain level of infection. For the germination speed index (GSI), no significant differences were found between seeds without inoculation (absence of fungi in the culture medium) and seeds inoculated with F. verticillioides for 48 and 96 hours. Seeds of the control treatment showed lower GSI compared to those of the other treatments. This result may be related to the fact that the seeds without inoculation or inoculated absorbed water from the culture medium, allowing a faster start of the germination process. In the health test, it was found that 100% of seeds were infected with F. verticillioides after 96 hours of inoculation (Table 2).
Ramos et al. (2014) found that the period of 16 hours was enough for F. graminearum and F. verticillioides to infect maize seeds. Similarly, Sousa et al. (2022) observed that inoculation times between 12 and 24 h were sufficient for considerable infection of F. verticillioides in maize seeds. However, it is worth mentioning that the longer the contact time of the seeds with the fungal colony, the greater the infection by the pathogen, causing a reduction in quality (Martín et al., 2022).
As the inoculation time increased from 48 h to 96 h, there was a reduction in the germination of seeds inoculated with A. flavus. In the evaluation of the germination speed index, no statistical differences were observed between seeds without inoculation and those inoculated with A. flavus. However, there was a significant difference compared to the control. In the health test, the highest percentage of infection by A. flavus was observed in seeds inoculated for 96 h (Table 2). Soybean seeds inoculated with Aspergillus ochraceus by water restriction also had their physiological potential reduced (Rocha et al., 2014). Morena et al. (2025) evaluated the effect of this fungus on maize seeds and observed that one of the important points is the use of alternative fungicides to avoid inducing resistance to commercial products such as tebuconazole.
In seeds inoculated with Penicillium sp., there was a decrease in germination after 48 h. Table 2 shows that there was a reduction in seed vigor by means of tetrazolium and GSI tests. Regarding the sanitary test, it is possible to verify that the incidence of Penicillium sp. did not differ in terms of inoculation times. Penicillium sp. is a storage fungus that, in addition to spoiling grains and seeds, produces mycotoxins that are highly toxic to humans, animals, and plants (Otero et al., 2020). Also showing low percentages of inoculum, this fungus has been reported to cause a reduction in the germination potential of peanut and sorghum seeds (Flávio et al., 2014). In addition to the use of commercial fungicides, Zacharias et al. (2024) also mention that one of the ways to mitigate Penicillium sp. in maize seeds may include alternative treatments such as coating the seeds with 2% chitosan and 0.30 g of glycerol g-1, which has shown promising results in reducing the occurrence of these fungi without harming the physiological potential of the seeds.
Identifying and quantifying differential gene expression may be a key factor in revealing the defense mechanisms of seeds against infection by F. verticillioides, Penicillium sp. and A. flavus. For each of the six comparisons, forward and reverse subtractive libraries were constructed, with the forward one enriched with cDNAs differentially expressed in healthy seeds and the reverse one enriched with cDNAs differentially expressed in contaminated seeds.
Regarding the genes expressed in the maize seeds in each of the subtractive libraries, it was possible to verify the expression of two, three, three, one, two and three coding genes in libraries 1, 2, 3, 4, 5 and 6, respectively (Table 3).
Genes expressed in maize seeds in each of the subtractive libraries indicating the access number; number of clones identified with the same annotation; E-value: identity with database sequences and number of clones. Name of the homologous ESTs of this sequence with the NCBI (National Center for Biotechnology Information - http://www.ncbi.nlm.nih.gov) database.
Protein-coding genes related to the beginning of germination were expected in libraries 1 to 3, while genes expressed through the presence of pathogens were expected in all libraries. Some clones of the maize genome were found, which may or may not be related to the presence of fungi in the seeds.
In general, the comparisons of gene expression intensity between the treatments (96, F, P and A) compared to the control (T), described below, are descriptive, based on the relative values presented in Figure 2. The six libraries constructed confirm the efficacy of the suppressive subtractive hybridization technique in the identification of candidate genes associated with the seed-pathogen interaction.
Expression of genes differentially expressed by qRT-PCR in maize seeds: control (T), without inoculation in culture medium for 96h (96) and inoculation of F. verticillioides (F), Penicillium sp. (P) and A. flavus (A).
In SSH 1 (T-96F), the Polyubiquitin and Zea mays clone 3516 hypothetical protein genes were protein coding (Table 3). There was a lower intensity in the expression of genes related to Polyubiquitin; however, the expression of this gene in seeds inoculated with F. verticillioides was slightly higher than in other treatments (Figure 2A). The low expression of polyubiquitin genes makes it difficult to understand the defense mechanisms of maize seeds against F. verticillioides, Penicillium sp. and A. flavus. There is little expression of polyubiquitin genes in most species, and these genes may differ in terms of nucleotide sequence and/or number of repeats of 228 nucleotides encoding the ubiquitin monomer (Han et al., 2021). Polyubiquitin accumulates in animal and plant cells after a stress response, such as heat shock (Han et al., 2021).
In SSH 1, the highest expression was that of the Zea mays clone 3516 hypothetical protein gene in seeds infected with F. verticillioides. The Zea mays clone 3516 hypothetical protein gene showed the highest peak of gene expression in this library and may represent a candidate marker associated with the interaction with F. verticillioides. Since the SSH 1 library compared seeds not subjected to water restriction (T) with seeds that were soaked and infected (96F), it is not possible to distinguish, from these data, whether the differential expression is due to fungal infection, the imbibition process, or a combination of both. Future studies with direct control of the imbibition condition are needed to confirm the specificity of this gene as a marker of infection.
From these results, it can be inferred that the protein expressed by the Zea mays clone 3516 hypothetical protein gene may play an important role in the response to the stress caused by the fungus. Therefore, further studies are needed to describe and characterize its function. Among the studies involving hypothetical proteins, those related to the following topics stand out: differential gene expression in the formation of sclerotia of Morchella conica (Chen et al., 2014), characteristics related to popcorn popping (Kaur et al., 2021), Bradyrhizobium japonicum symbiosis (Ferreira et al., 2022), storage proteins responsible for bruchid resistance in common bean seeds (Maro et al., 2022) and identification of QTL for protein contents in peanut seeds (Chen et al., 2024).
In SSH 2 (T-96P), the differential expression between maize seeds treated with fungicides and maize seeds inoculated with Penicillium sp. was verified (Figure 2B). The genes identified and differentially expressed were: Aspartate aminotransferase, Zea mays zinc finger protein and Zea mays protein SPATULA. Reduced expression of the Aspartate aminotransferase and Zea mays zinc finger protein genes can be observed regardless of treatment. Cytosolic and mitochondrial isozymes of Aspartate Aminotransferase have function in the photosynthetic cycle in NAD-malic enzymes of C4 plants and are expressed at elevated levels in mesophyll cells and bundle sheath cells, respectively (Shah et al., 2023). Zinc finger protein, on the other hand, is a gene that encodes hydrophilic proteins and is related to dehydration (Agarwal and Khurana, 2020).
The highest expression levels in SSH 2 were those of the Zea mays protein SPATULA gene in seeds inoculated with F. verticillioides and A. flavus. It is also possible to verify that the highest expression occurred in seeds inoculated with A. flavus. It is worth noting, however, that this difference may be associated not only with fungal infection, but also with the absence of water restriction in the control (T), which limits the attribution of expression exclusively to the pathogen. According to Martínez-Estrada et al. (2025), the Spatula gene (SPT) encodes a bHLH transcription factor present in fungi, plants and animals, which promotes the growth and development of tissues resulting from the carpel margins, including the septum and the transmitting tract. This gene is also linked to the repression of seed germination, being a potent regulator of cotyledon expansion and inhibiting leaves (Liang and Hu, 2024).
The differential expression between maize seeds treated with fungicides and maize seeds inoculated with Aspergillus flavus was verified in SSH 3 (T-96A) (Figure 2C). The genes expressed were Glyceraldehyde-3-phosphate dehydrogenase, Alcohol dehydrogenase (ADH) and a Sorghum bicolor hypothetical protein. The expressions of the genes ADH and Sorghum bicolor hypothetical protein were lower. The Glyceraldehyde-3-phosphate dehydrogenase gene linked to stress caused by A. flavus showed higher expression, and this gene may be a candidate marker for the detection of A. flavus infection in maize seeds, although the absence of control for the imbibition condition in the SSH 3 library (T versus 96A) limits this interpretation. It is also worth mentioning that this gene is expressed in dry seeds (96), allowing early selection without the need to germinate the seeds.
Glyceraldehyde-3-phosphate dehydrogenase is a cytosolic developmental gene in maize seeds, encoded by a small multi-gene family consisting of gpc1, gpc2, gpc3 and gpc4. GPC3/4 proteins are synthesized in roots during anoxic conditions and are known to be one of the polypeptides of anaerobic pathways (Sanclemente et al., 2021). ADH is a relevant enzyme, as it converts acetaldehyde into ethanol, a compound with lower toxicity and that reduces the speed of the deterioration process (Xu et al., 2019). Thus, the seeds are less susceptible to the deleterious action of acetaldehyde with the higher activity of ADH (Cavalcante et al., 2023).
In SSH 4, 5 and 6 (Figures 1D, 1E, 1F), the presence of genes encoding proteins related to germination was not expected, since the control (“driver”) was subjected to the same conditions of imbibition as the treatments (“tester”), with the objective of canceling this difference. In SSH 4 (96-96F), there was differential expression between maize seeds without inoculation, kept in culture medium for 96 hours (96), and maize seeds inoculated with F. verticillioides (F). The expression of the protein Anthranilate synthase can be observed, which is a defense metabolite, whose accumulation is promoted by the ZmPep1 gene (ortholog of Arabidopsis elicitor peptide 1). Ortiz-Morea and Reyes-Bermudez (2019) concluded that peptides belonging to the Pep family have the function of endogenous regulator of innate immunity in plants and may have the potential to improve disease resistance in crops.
In SSH 5 (96-96P), the differential expression between maize seeds without inoculation, kept in culture medium for 96 hours (96), and maize seeds inoculated with Penicillium sp. was studied. (P). The exclusive genes for this library were Cysteine Proteinase and the isoenzyme Catalase B. It is possible to verify that the seeds of the control (T) also showed the expression of these genes and there was specificity for the detection of Penicillium sp. Cysteine Proteinase is related to pathophysiological processes, and its higher expression is probably involved with fungal infection (Yang et al., 2023). Yuan et al. (2024) found that cysteine is related to the germination process and concluded that CCP1 mRNA is expressed in maize seeds. Catalase (CAT) is also an important marker, as it is an enzyme that converts peroxide (H2O2) into H2O and O2, playing a key role in protection and repair systems during deterioration (Catão et al., 2020). In this context, the decrease in their expression can make maize seeds more sensitive to the effects of reactive oxygen species (ROS), free radicals, unsaturated fatty acids in the membrane and peroxidation products of secondary lipids (Catão et al., 2020; Pinheiro et al., 2023).
In SSH 6 (96-96A), the differential expression was verified between maize seeds without inoculation, kept in culture medium for 96 hours (96), and maize seeds inoculated with A. flavus (A). The Glyceraldehyde-3-phosphate dehydrogenase gene was also expressed in this library, as well as in SSH 3. With this finding, it can be inferred that this gene is associated with A. flavus infection. The Zea mays clone 276247 hypothetical protein and Superoxide dismutase 4 genes were also expressed. Superoxide dismutase (SOD) is an enzyme that plays an important role in the response to oxidative stress in plants, acting on the O2 - radical, forming hydrogen peroxide and molecular oxygen. SOD is an important marker that helps in the detection of processes related to seed deterioration by biotic or abiotic factors (Catão et al., 2020; Pinheiro et al., 2023).
In summary, the results obtained in the physiological and gene expression analyses of this study reinforce the complexity involved in the detection of seeds infected with F. verticillioides, Penicillium sp. and A. flavus. This complexity emphasizes the need for studies that help in the development of methods that allow faster selection of infected materials, reducing the work and time required for such selection, becoming an important tool.
CONCLUSIONS
The physiological and sanitary quality of maize seeds is compromised by the infection by F. verticillioides, A. flavus and Penicillium sp. The suppressive subtractive hybridization technique proved to be efficient in detecting differentially expressed genes associated with this interaction, with anthranilate synthase, cysteine proteinase, catalase B, glyceraldehyde-3-phosphate dehydrogenase and superoxide dismutase 4 standing out as potential markers more directly related to fungal infection, while the genes of the T versus 96F/96P/96A libraries should be interpreted as candidates, subject to confirmation in assays with control of imbibition.
REFERENCES
-
ABREU, J.R.; PAIVA, L.V.; RODRÍGUEZ, M.A.D.; SILVA, A.T.; HENRIQUES, A.R.; CHALFUN-JUNIOR, A. Identification and quantification of differentially expressed genes associated with citrus blight (Citrus sp.). Ciência e Agrotecnologia, v.39, n.1, p.32-38, 2015. https://doi.org/10.1590/S1413-70542015000100004
» https://doi.org/https://doi.org/10.1590/S1413-70542015000100004 -
AGARWAL, P.; KHURANA, P. TaZnF, a C3HC4 type RING zinc finger protein fromTriticum aestivumis involved in dehydration and salinity stress.Journal of Plant Biochemistry and Biotechnology, v.29, p.395-406, 2020. https://doi.org/10.1007/s13562-019-00546-8
» https://doi.org/https://doi.org/10.1007/s13562-019-00546-8 -
AKHTAR, J.; KUMAR, P. Seed-borne fungi: challenges in seed health testing for biosecurity and agricultural sustainability.Indian Phytopathology, v.77, n.2, p.257-269, 2024. https://link.springer.com/article/10.1007/s42360-024-00723-3
» https://link.springer.com/article/10.1007/s42360-024-00723-3 - BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Manual de Análise Sanitária de Sementes Brasília-DF, 2009. 202p.
- BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Regras para Análise de Sementes Brasília: MAPA, 2025.
-
CATÃO, H.C.R.M.; CAIXETA, F.; LOPES, A.M.; NERY-SILVA, F.A.; SÁ-JÚNIOR, A. Antioxidant activity and physiological performance of popcorn seed after saline stress and analysis of seedling images. Ciência e Agrotecnologia , v.44, e005020, 2020. https://doi.org/10.1590/1413-7054202044005020
» https://doi.org/https://doi.org/10.1590/1413-7054202044005020 -
CAVALCANTE, J.A.; GADOTTI, G.I.; PINHEIRO, R.M.; SILVA, R.N.O.; OLIVEIRA, F.K.; MORAES, D.M. Vigor and anaerobic metabolism of soybean seeds evaluated by ethanol test. Journal of Seed Science, v.45, e202345007, 2023. https://doi.org/10.1590/2317-1545v45263825
» https://doi.org/https://doi.org/10.1590/2317-1545v45263825 -
CHANDRASEKARAN, M.; RAMAN, C.; KARTHIKEYAN, K.; PARAMASIVAN, M. Functional annotation of hypothetical proteins derived from suppressive subtraction hybridization (SSH) analysis shows npr1 (non-pathogenesis related) - like activity.Agronomy, v.9, n.2, p.57, 2019. https://doi.org/10.3390/agronomy9020057
» https://doi.org/https://doi.org/10.3390/agronomy9020057 -
CHEN, H.; LIU, N.; HUANG, L.; HUAI, D.; XU, R.; CHEN, X.; GUO, S.; CHEN, J.; JIANG, H. Identification of a major qtl for seed protein content in cultivated peanut (Arachis hypogaeaL.) using QTL-Seq.Plants, v.13, n.17, 2368, 2024. https://doi.org/10.3390/plants13172368
» https://doi.org/https://doi.org/10.3390/plants13172368 -
CHEN, L.J.; CHAI, H.M.; CHEN, W.M.; HUANG, X.Q.; ZHAO, Y.C. Gene expressing difference in sclerotial formation ofMorchella conicaIndian Journal of Microbiology, v.54, p.274-283, 2014. https://doi.org/10.1007/s12088-014-0445-0
» https://doi.org/https://doi.org/10.1007/s12088-014-0445-0 -
DE PALMA, M.; D’AGOSTINO, N.; PROIETTI, S.; BERTINI, L.; LORITO, M.; RUOCCO, M.; CARUSO, C.; CHIUSANO, M.L.; TUCCI, M. Suppression subtractive hybridization analysis provides new insights into the tomato (Solanum lycopersicum L.) response to the plant probiotic microorganism Trichoderma longibrachiatum MK1. Journal of Plant Physiology, v.190, p.79-94, 2016. https://doi.org/10.1016/j.jplph.2015.11.005
» https://doi.org/https://doi.org/10.1016/j.jplph.2015.11.005 -
FERREIRA, E.G.C.; GOMES, D.F.; DELAI, C.V.; BARREIROS, M.A.B.; GRANGE, L.; RODRIGUES, E.P.; HENNING, L.M.M.; BARCELLOS, F.G.; HUNGRIA, M. Revealing potential functions of hypothetical proteins induced by genistein in the symbiosis island ofBradyrhizobium japonicumcommercial strain SEMIA 5079 (= CPAC 15).BMC Microbiology, v.22, p.122, 2022. https://doi.org/10.1186/s12866-022-02527-9
» https://doi.org/https://doi.org/10.1186/s12866-022-02527-9 -
FLÁVIO, N.S.D.S.; SALES, N.L.P.; AQUINO, C.F.; SOARES, E.P.S.; AQUINO, L.F.S.; CATÃO, H.C.R. M. Health and physiological quality of sorghum seeds treated with aqueous extracts and essential oils.Semina: Ciências Agrárias, v.35, n.1, p.7-20, 2014. https://doi.org/10.5433/1679-0359.2014v35n1p7
» https://doi.org/https://doi.org/10.5433/1679-0359.2014v35n1p7 - FRANÇA-NETO, J.B.; KRZYZANOWSKI, F.C.; DIAS, M.C.L.L.; BARROS, A.S.R. Teste de tetrazólio em sementes de milho. In: KRZYZANOWSKI, F.C.; VIEIRA, R.D.; FRANÇA-NETO, J.B, MARCOS-FILHO, J. (Eds.). Vigor de sementes: conceitos e testes Londrina: ABRATES, 2020. p.501-517.
-
HAN, S.W.; JUNG, B.K.; RYU, K.Y. Regulation of polyubiquitin genes to meet cellular ubiquitin requirement. BMB Reports, v.54, n.4, p.189-195, 2021. https://doi.org/10.5483/BMBRep.2021.54.4.005
» https://doi.org/https://doi.org/10.5483/BMBRep.2021.54.4.005 -
KAUR, S.; RAKSHIT, S.; CHOUDHARY, M.; DAS, A.K.; KUMAR, R.R. Meta-analysis of QTLs associated with popping traits in maize (Zea maysL.). Plos One, v.16, n.8, e0256389, 2021. https://doi.org/10.1371/journal.pone.0256389
» https://doi.org/https://doi.org/10.1371/journal.pone.0256389 -
LAMICHHANE, J.R.; YOU, M.P.; LAUDINOT, V.; BARBETTI, M.J.; AUBERTOT, J.N. Revisiting sustainability of fungicide seed treatments for field crops. Plant Disease, v.104, n.3, p. 610-623, 2020. https://doi.org/10.1094/PDIS-06-19-1157-FE
» https://doi.org/https://doi.org/10.1094/PDIS-06-19-1157-FE -
LIANG, L.; HU, X. Spatula as a versatile tool in plant: the progress and perspectives of spatula (spt) transcriptional factor. Phyton-International Journal of Experimental Botany, v.93, n.3, p.517-531, 2024. https://doi.org/10.32604/phyton.2024.049277
» https://doi.org/https://doi.org/10.32604/phyton.2024.049277 - MACHADO, J.C. Patologia de Sementes: fundamentos e aplicações Brasília DF. MEC/ESAL/FAEPE, 2000. 107p.
-
MAGUIRE, J.D. Speed of germination - aid in selection and evaluation for seedling emergence and vigor. Crop Science, v.2, n.2, p.176177, 1962. https://doi.org/10.2135/cropsci1962.0011183X000200020033x
» https://doi.org/https://doi.org/10.2135/cropsci1962.0011183X000200020033x -
MARO, C.N.; MASSAWE, D.P.; TRYPHONE, G.M.; MYERS, J.R.; DAVIS, J.W.; KUSOLWA, P.M. Identification of potential seed storage protein responsible for bruchid resistance in common bean landraces from Tanzania and Malawi. African Journal of Biotechnology, v.21, n.1, p.35-45, 2022. https://doi.org/10.5897/AJB2021.17354
» https://doi.org/https://doi.org/10.5897/AJB2021.17354 -
MARTÍN, I.; GÁLVEZ, L.; GUASCH, L.; PALMERO, D. Fungal pathogens and seed storage in the dry state.Plants, v.11, n.22, 3167, 2022. https://doi.org/10.3390/plants11223167
» https://doi.org/https://doi.org/10.3390/plants11223167 -
MARTÍNEZ-ESTRADA, E.; BERNAL-GALLARDO, J.J.; LÓPEZ-GÓMEZ, P.; MORA-FRANCO, D.; CELSO-ESPINOZA, M.; GUERRERO-ESPERANZA, M.; DÍAZ-RAMÍREZ, D.; MARSCH-MARTÍNEZ, N.; ORDAZ-ORTIZ, J.J.; FOLTER, S. Conserved and novel roles of the bHLH transcription factor SPATULA in tomato. Journal of Experimental Botany, v.76, n,21, eraf029, 2025. https://doi.org/10.1093/jxb/eraf029
» https://doi.org/https://doi.org/10.1093/jxb/eraf029 -
MORENA, C.; ACCINELLI, C.; BRUNO, V.; ABBAS, H.K.; PAULK, R.T.; SHIER, W.T. Induced selection of tebuconazole-resistant Aspergillus flavus isolates during germination of treated corn seeds. Journal of Agriculture and Food Research, v.19, 101566, 2025. https://doi.org/10.1016/j.jafr.2024.101566
» https://doi.org/https://doi.org/10.1016/j.jafr.2024.101566 -
ORTIZ-MOREA, F.A.; REYES-BERMUDEZ, A.A. Endogenous Peptides: Key Modulators of Plant Immunity. In: JOGAIAH, S., ABDELRAHMAN, M. (Eds.) Bioactive Molecules in Plant Defense Springer, Cham. 2019. https://doi.org/10.1007/978-3-030-27165-7_10
» https://doi.org/https://doi.org/10.1007/978-3-030-27165-7_10 -
OTERO, C.; ARREDONDO, C.; ECHEVERRÍA-VEGA, A.; GORDILLO-FUENZALIDA, F. Penicillium sp. mycotoxins found in food and feed and their health effects. World Mycotoxin Journal, v.13, n.3, p.323-343, 2020. https://www.wageningenacademic.com/doi/abs/10.3920/WMJ2019.2556
» https://www.wageningenacademic.com/doi/abs/10.3920/WMJ2019.2556 -
PFAFFL, M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research, v.29, n.9, p.2002-2007, 2001. https://doi.org/10.1093/nar/29.9.e45
» https://doi.org/https://doi.org/10.1093/nar/29.9.e45 -
PINHEIRO, D.T.; DIAS, D.C.F.S.; SILVA, L.J.; MARTINS, M.S.; FINGER, F.L. Oxidative stress, protein metabolism, and physiological potential of soybean seeds under weathering deterioration in the pre-harvest phase.Acta Scientiarum Agronomy, v.45, e56910, 2023. https://doi.org/10.4025/actasciagron.v45i1.56910
» https://doi.org/https://doi.org/10.4025/actasciagron.v45i1.56910 -
R CORE TEAM. R: A language and environment for statistical computing R Foundation for Statistical Computing, Vienna, Austria. 2025. https://www.R-project.org/
» https://www.R-project.org/ -
RAMOS, D.P.; BARBOSA, R.M.; VIEIRA, B.G.T.L.; PANIZZINI, R.C.; VIEIRA, R.D. Infecção por Fusarium graminearum e Fusarium verticillioides em sementes de milho. Pesquisa Agropecuária Tropical, v.44, n.1, p.24-31, 2014. http://dx.doi.org/10.1590/S1983-40632014000100011
» https://doi.org/http://dx.doi.org/10.1590/S1983-40632014000100011 -
ROCHA, F.D.S.; CATÃO, H.C.R.M.; BRANDÃO, A.D.A.; GOMES, L.A.A. Damage caused by different inoculum levels of Aspergillus ochraceus on the vigour of soybean seeds. Semina: Ciências Agrárias , v.35, n.6, p.2895-2903, 2014. http://dx.doi.org/10.5433/1679-0359.2014v35n6p2895
» https://doi.org/http://dx.doi.org/10.5433/1679-0359.2014v35n6p2895 - SAMBROOK, J.; FRITSCH, E.F.; MANIATIS, T. Molecular Cloning - A Laboratory Manual (2a Ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. 1989.
-
SANCLEMENTE, M.A.; MA, F.; LIU, P.; DELLA PORTA, A.; SINGH, J.; WU, S.; COLQUHOUN, T.; JOHNSON, T.; GUAN, J-C.; E KOCH, K. Sugar modulation of anaerobic-response networks in maize root tips.Plant Physiology, v.185, n.2, p.295-317, 2021. https://doi.org/10.1093/plphys/kiaa029
» https://doi.org/https://doi.org/10.1093/plphys/kiaa029 -
SHAH, W.H.; RASOOL, A.; PADDER, S.A.; SINGH, R. K.; PRASAD, M.; TAHIR, I.; REHMAN, R.; HAKEEM, K. R. Decarboxylation mechanisms of the C4 cycle in foxtail millet observed under salt and selenium treatments.Plant Growth Regulation, v.99, p.65-83, 2023. https://doi.org/10.1007/s10725-022-00888-9
» https://doi.org/https://doi.org/10.1007/s10725-022-00888-9 -
SOUSA, R.R.D.; OSÓRIO, P.R.A.; NOSÉ, N.P.E.; ARRUDA, G.L.D.; FERREIRA, T.P.D.S.; HAESBAERT, F. M.; SANTOS, G.R.D. Detection and transmission of Fusarium verticillioides in corn seeds according to the plant stage. Acta Scientiarum. Agronomy, v.44, e53213, 2022. https://doi.org/10.4025/actasciagron.v44i1.53213
» https://doi.org/https://doi.org/10.4025/actasciagron.v44i1.53213 -
XU, X.; NIU, C.; LIU, C.; LI, Q. Unraveling the mechanisms for low-level acetaldehyde production during alcoholic fermentation inSaccharomyces pastorianusLager Yeast. Journal of Agricultural and Food Chemistry, v.67, p.2020-2027, 2019. https://doi.org/10.1021/acs.jafc.8b06868
» https://doi.org/https://doi.org/10.1021/acs.jafc.8b06868 -
YANG, N.; MATTHEW, M.A.; YAO, C. Roles of cysteine proteases in biology and pathogenesis of parasites.Microorganisms, v.11, n.6, 1397, 2023. https://doi.org/10.3390/microorganisms11061397
» https://doi.org/https://doi.org/10.3390/microorganisms11061397 -
YUAN, W.; CHEN, X.; DU, K.; JIANG, T.; LI, M.; CAO, Y.; LI, X.; DOEHLEMANN, G.; FAN, Z.; ZHOU, T. NIa-Pro of sugarcane mosaic virus targets Corn Cysteine Protease 1 (CCP1) to undermine salicylic acid-mediated defense in maize.PLoS Pathogens, v.20, n.3, e1012086, 2024. https://doi.org/10.1371/journal.ppat.1012086
» https://doi.org/https://doi.org/10.1371/journal.ppat.1012086 -
ZACHARIAS, M.B.; FORTI, V.A.; SILVA, M.A.D. Physiological potential and health quality of corn seeds coated with chitosan. Scientia Agricola, v.81, e20230121, 2024. https://www.scielo.br/j/sa/a/9vKsnYRPxYCyPtNmCH9Xtqz/?lang=en
» https://www.scielo.br/j/sa/a/9vKsnYRPxYCyPtNmCH9Xtqz/?lang=en
