Evaluation of symptoms and histological analysis
The characteristic spots of Black Sigatoka are yellow in color, which, over time and the evolution of the pathogen, become brown, grow, and aglutinate, inducing tissue death. The spots in proportion that increase become necrotic, impairing plant photosynthesis (Borah et al. 2022).
Ascospores are produced in necrotic tissues, completing their cycle in the necrotrophic phase (Favreto; Model; Tonietto 2007), however, this statement goes against what was observed in our study. The presence of ascospores in the treatments with water and 8% glycerol occurred in non-necrotic leaves. This type of result could be expected in plants treated with 8% glycerol because the high level of this compound, which consequently has a higher amount of carbon, was acting more as an energy source for the fungus (Chanda et al. 2008).
M. fijiensis, as a hemibiotrophic fungus, has a biotrophic cycle that lasts 3-4 weeks without causing death to the plant (AGRIOS 2019), which explains the way the symptoms occurred during the 5 weeks of evaluation, not reaching the necrosis phase. Treatment with 8% glycerol showed ascospores and greater severity of symptoms. This result again suggests Glycerol possibly acting as a carbon source for the fungus, corroborating the results obtained for ID and AACPD analysis.
Zhang et al. (2015) verified cell death in plants treated with higher doses of Glycerol in the cocoa crop treated with doses above 300 mM, where chlorosis and cell death were visualized at the tips of the leaves. In banana, cultivar Grande Naine, no significant phenotypic differences were seen in plants treated with 3, 5 and 8% Glycerol compared to untreated plants.
From the clarification, it was possible to visualize several stages of development of black Sigatoka, from hyphae to spores of sexual reproduction in germinated and non-germinated forms. It has been scientifically proven that the higher the density of stomata, where the fungus M. fijiensis infects the plant, the greater the infection. Grande Naine, a susceptible genotype, has a high density of stomata, which facilitates the penetration of the fungus (SOARES et al., 2022). The plant, with physical and chemical defense responses, uses mechanisms to try to defend itself from the invader. The cell wall restricts the fungal germ tube and biochemical defense is triggered by ITP and ITE by the production of reactive oxygen species and others. Secondary metabolites also act as molecules in the development of acquired systemic resistance (SAR) (Kaur et al. 2022).
Plants treated with 8% Glycerol showed similar results to water-treated plants, with some fungal structures in the first hours of evaluation. In the treatment with 5% glycerol, the first visualization of conidia was at 5 DAI, while in the same period the treatment with 8% glycerol presented ungerminated ascospores. Initially, treatment with 3% glycerol demonstrated a higher level of resistance, developing structures from 15 DAI. Li et al. (2016) identified that the higher the dose of Glycerol (1-4%), the greater the resistance developed by wheat plants against the oidium, but considered the best dose at 3% because 4% glycerol presents cell death at the tips of the leaves.
Li et al. (2020) found spore germination in ~90% of water-treated plants, suggesting that Glycerol induced resistance against the wheat fungal pathogen powdery mildew. This may be related to the present study in which structures were visualized in the control treatment from the first days and in the treatment with a higher dose of Glycerol, 8%.
For wheat, different times of glycerol application were evaluated, before and after inoculation, and it was proven that the best time to start the treatment is 1-2 days before inoculation. Plants treated with Glycerol were highly resistant, while those treated with water only were susceptible (Li et al. 2016).
Gene expression
The glycerol-mediated induction of defense in plants is related to the increase in glycerol-3-phosphate (G3P) and the decrease in oleic acid (18:1). Glycerol can be used in plants to induce resistance against pathogens, but has even been considered toxic in plants when used in high concentrations (Zhang et al. 2015).
The study of gene expression in banana plants infected by the M. fijiensis isolate contributes to the identification of the beginning of plant defense, since visually this was not possible due to the initial phase being biotrophic and not presenting symptoms initially (Rodríguez et al. 2016). In the study performed here, it was possible to identify that the signaling gene was expressed early on in the infection.
In a study with resistant and susceptible varieties, Rodríguez et al. (2016) were able to identify several defense genes in the resistant genotype, Calcutta 4, and different induction in the susceptible one, Williams. In Calcutta 4, induction was early, as in the present study; in susceptible plants, there was gene induction at the beginning of infection.
A transduction signal developed by plants after glycerol treatment in response to the pathogen may be salicylic acid, an important hormone for acquired systemic resistance (Xá et al. 2001) and oleic acid (Mendel et al.2012); its response is dose-dependent (Zhang et al. 2015). This explains the induction of the STRANS and KINLRR genes in banana plants, the higher the glycerol dose, the higher the expression of these genes. Signal transduction is activated when the plant recognizes the presence of the pathogen. When recognizing, it releasesCa2+, considered the main mediator against plant stress (Ding et al. 2022).
The KINLRR gene is a leucine-rich kinase gene. This gene acts as a receptor that detects abnormalities created by biotic and abiotic factors in plants (Senseverino et al. 2010; Antolín-Loovera, Binder and parniske 2012). Leucine-rich proteins are effector-triggered immunity (ETI). The ETI is activated after the pathogen manages to overcome the host's first line of defense, acting as a second line of defense (Noman, Aqeel, Lou 2019).
Signal transduction in plants involves several lines of plant defense to prevent the development of the pathogen, where the responses are the creation of reactive oxygen species (ROS), pathogenesis-related genes, callose deposition, salicylic acid, and mitogen-activated protein kinase (MAPK) signaling, among others (Liu and Lam 2019). Hormones are molecules that act on the plant in various physiological processes, including plant defense. Among the main ones, jasmonic acid, salicylic acid, ethylene, abscisic acid, auxin, gibberellin stand out (Shigenaga and Argueso 2016).
Treatment with 5% glycerol had long-term positive responses for the two genes tested. STRANS gene expression in this treatment remained high at 60 DAI, while for KINLRR, it decreased compared to 30 DAI. Comparing treatments 5 and 8% glycerol, the treatment that had the most gene expression, 5% glycerol, maintained this result until the end of the evaluations.
The way Glycerol acts in banana plants still needs several studies to prove, but the increase in G3P and the decrease in oleic acid occurs through the acylation between the two in plasmids, the elevation of G3P contributes to signaling through salicylic acid (AS) and jasmonic acid (AJ) (Kachroo et al. 2004). Ethylene is also part of signaling, and each hormone activates a pathway individually, together, or antagonistically, but each response depends on the plant x pathogen interaction (Chandra-shekara et al. 2007). Related to plant defense against pathogens, induction against necrotrophic microorganisms occurs through JA, the hormone responsible for defense against necrotrophic pathogens (Matric et al. 2016).
LRR is an effector recognition gene that causes virulence proteins to become avirulent, it has a second line of defense known as ETI, which was previously called gene-to-gene or race-specific resistance (Jones and Dangl 2006). LRR induces resistance in plants attacked by biotrophic or hemibiotrophic microorganisms, that is, organisms that allow the plant to remain alive for at least one period (Glazebrook, 2005), such as the case of the fungus M. fijiensis, which is a hemibiotrophic fungus.
The KINLRR gene was expressed during the first hour of evaluation during the interaction of Musa spp. x M. fijiensis, demonstrating that the expression of this gene was rapid in the detection of stress, recognizing the presence of the pathogen in only 1 HAI and induced resistance in most of the evaluated times (Oliveira 2020).
Signal transduction acts on the plant when pathogens invade it. The signals cooperate for resistance with the activation of molecules such as salicylic acid, jasmonate, and ethylene, which are considered precursors of acquired systemic resistance (Yang, Shah, and Klessig 1997; Kachroo et al. 2004). With signal transduction, plant defense mechanisms are activated and the pathogen's development is impaired (Dangue and Jones 2001). Mandel et al. (2012) point out that these signals, such as nitric oxide, are involved in the oleic acid pathway (18:1). Not only are hormones part of the signal transduction pathway, but also reactive species and pending calcium signaling, and these can act in interaction (Tiwari 2018).
In wheat culture, Glycerol induced plant hormone signal transduction through auxin, PP2C and Jasmonate Zim Domain (JAZ). The positive induction of JA by Glycerol could contribute to wheat resistance to the fungus of Oide (Li et al. 2020).
For the target gene KINLRR, the positive expression with the dose of 5% glycerol was decreasing in the final moments of the evaluations, which can be deduced that more applications of this compound are necessary during the period of infection for the plant to maintain a level of resistance without so many oscillations in the expressions of these resistance genes.
In the present study, the regression result, together with the gene expression data, demonstrated a tendency of the cultivar Big Naine to respond more resistantly to M. fijiensis isolate at a concentration of 5% glycerol. This result differs from responses with other plants in which the 3% dose was the most expressed resistance to plants (LI et al. 2016; LI et al. 2020; Gazolla et al. 2019).
Histochemistry
This is the first study on the Musa spp. x M. fijiensis interaction in response to Glycerol, as well as the first to use histochemistry to visualize defense mechanisms for the defined treatments. The detection of callose in the first days after inoculation indicates that the application of Glycerol led to a tendency to condition the plant to a state of resistance to fungal attack. Soares et al. (2022) did not identify the presence of callose in banana plants in susceptible genotypes Grande Naine and Akondro Mainty infected with Black Sigatoka, evaluated up to 21 Dai.
Callose is a beta-1,3-glucan polysaccharide that is present in the cell wall of several plants, aiding in development and contributing as a defense response against biotic and abiotic factors (Chen and Kim, 2009). As a structural mechanism, it provides cell wall resistance as a form of containment to prevent pathogen penetration, thus preventing associations with each other (Stangarlin et al. 2011).
As it is a pioneering work, it is believed that the use of Glycerol to control SN in bananas of the cultivar Grande Naine seems promising. However, further studies involving more applications of Glycerol prior to inoculation, as well as the use of a greater number of genes in the gene expression study, will be able to respond to the responses more robustly. The possibility of using this compound in banana plantations of cultivar GN will bring great benefits to the production chain of the crop, with great impact on the environment and human health.