Wen 丨 Nameless Hao
Editor丨Nameless Hao
Fusarium graminears is one of the most virulent pathogens that attacks wheat plants and significantly reduces global wheat yields.
The current work aims to elucidate the interaction between two initiation solutions, calcium (Ca) or salicylic acid (SA)), and Fusarium graminis inoculation, depending on the growth rate, physiological properties, and molecular response of wheat seedlings.
In potting experiments, wheat kernels were germinated using pre-inoculated Fusarium graminus or inoculum sand soil after 12 h of priming in distilled water, 5-mMCaCl2, or 0.05-mMSA.
The results showed that Fusarium graminus inoculation reduced growth rate and chlorophyll content, but promoted carotenoids, stress markers (electrolyte leakage, lipid peroxidation, protein oxidation, hydrogen peroxide, and hydroxyl radicals), antioxidant molecules (AsA, phenols, and flavonoids), penetrants (GB, amino acids, and proline), and antioxidant enzymes (CAT, GPX, SOD, PPO, and PAL).
In addition to these, fungal infections enhance the expression of CAT, GR, PR4, MT, and PCS genes, however pre-soaking wheat kernels in Ca or SA solution helps restore growth rate, chlorophyll content, and antioxidant capacity, thereby mitigating the negative effects of fungal inoculation.
It can also reduce induced oxidative stress and downregulate gene expression in Fusarium graminin-inoculated wheat seedlings, and by minimizing the negative effects of Fusarium graminis infestation, we can use Ca or SA initiation to properly stimulate growth and readjust the oxidative state of wheat seedlings.
Effects of coercion
On the basis of the oxidative state, when plants are subjected to biological stress, which has an impact on the agricultural sector and poses a threat, fungal infections are a constant threat to crop production and food security.
Fusarium is one of the most common fungal infections in small grains and causes extensive damage, with gibberella (FHB), seedling blight and crown rot, and foot brown rot (FCFR) being the most important diseases of wheat caused by Fusarium gramines.
Therefore, climate change, reduced tillage, crop rotation or the habit of throwing straw into the field may lead to an increase in FHB cases, and Fusarium graminus is listed as one of the top ten plant fungal pathogens based on FHB severity and fungal toxicity.
After wheat head infection and colonization, Fusarium graminus interferes with grain development and contaminates the surviving kernels with mycotoxin mixtures, making them unsuitable for human and feed utilization, impairing overall wheat yields.
The most popular mycotoxins in Fusarium cereals include deoxynivalenol, fusarium and Fusarium C, of which deoxynivalenol is the most important virulence factor for Fusarium graminformin.
However, by manipulating cytoplasmic calcium concentrations to enable plant development and response as key players, plants have developed effective mechanisms for perceiving, translocating, and responding to various biotic and abiotic stimuli.
But calcium (Ca2+) is a versatile endogenous second messenger that triggers multiple responses involved in growth, defense, and adaptation to biotic and abiotic stimuli in multiple signaling pathways.
In addition, Ca2+ is a tightly regulated ion in the cellular compartment, and its spatial and temporal control of concentration makes it a versatile signaling component in plants.
Calcium ions have also been shown to play a role in the crosslinking of plant cell walls and mid-layer pectin polysaccharides, enhancing them and promoting tissue antifungal enzyme activity.
In addition, the role of plant hormones in reducing the harmful consequences and effects of biological and environmental stressors, salicylic acid (SA) is one of the plant hormones that acts as a signaling and regulatory molecule in plant responses to various stimuli by regulating metabolic and molecular processes.
Thus, seed germination, stomatal closure, ion absorption and transport, membrane permeability, photosynthesis, and plant development have all been shown to be affected by exogenous application of SA.
According to numerous studies, SA is an important phenylpropane compound that regulates plant tolerance to pathogenesis and other stress conditions.
In plants, when a pathogen encounters a plant organ, the phenomenon of systemically acquired resistance (SAR) occurs, causing a local hypersensitivity reaction and then transmitting the signal to the surrounding plant organ, preventing additional infection.
These systemic responses are mainly based on hormonal crosstalk and small molecule interactions to induce broad-spectrum drug resistance, and salicylate-mediated signaling triggers SAR.
The salicylic acid response is associated with the expression of pathogenesis-related proteins (PRPs), one of which is PRP1, a salicylic acid marker that is upregulated during SA reactions in many plants.
Water and salicylic acid is an important plant defense hormone that helps plants defend against a variety of biotrophic and semi-biotrophic lifestyle diseases.
Increased SA concentrations at pathogen sites of attack can trigger H2O2 formation, leading to allergic reactions and lignification of plant cell walls at the site of pathogen infection, thereby promoting disease resistance.
In addition, we found that the treatment of SA improved the resistance of wheat to Fusarium graminearum infection by regulating antioxidant pathways, thereby increasing H2O2 concentration and promoting pathogen-related genes.
However, the correlation between H2O2 and SA levels confirms the interaction between these two signals in enhancing plant defenses, as they are closely related to the activity level of PRP.
Molecular effects on wheat
Wheat was listed as the most basic agricultural product in 2014, with production exceeding 220 million hectares and producing 730 million tonnes. In many regions, it is used as the main staple food.
Bread, for example, is considered the richest source of dietary protein in human nutrition, accounting for the fifth largest total dietary protein globally.
In addition to the main components of proteins, carbohydrates, and lipids, wheat kernels are rich in phytochemicals, vitamins, antioxidants, and macro and micronutrients.
In previous studies, we described how penetrant molecules such as glycine betaine, proline, and free amino acids help plants cope with invading microorganisms through a variety of mechanisms.
These include regulating cell osmotic pressure, ROS lysis, maintaining membrane fluidity, and stabilizing cell structure, and based on the exploration of synthesis, it is unclear exactly how SA works to reduce the pathogenicity of Fusarium graminus in wheat.
And to our knowledge, no studies have been conducted to determine the role of Ca in resistance to this pathogen, so the main goal of this study is to explore whether initiation of CA or SA can indeed mitigate the effect of inoculation with Fusarium graminis on wheat seedlings and yields.
Next, we will focus on the growth rate, metabolic activity, antioxidant status, and molecular reactivity at the seedling stage.
Seedlings and fungal isolates
The wheat grains were purchased from the Agricultural Research Centre in Gemisa, Egypt, and selected for apparent size and morphological homogeneity, and the Centre for Mycology at Assiut University contributed Fusarium graminaceum Schwabe for this study.
According to botanist Sobi, Fusarium graminus spores are inoculated into the soil using sand corn flour (SCM) medium.
After 7 days of growth of fungal mycelium on potato glucose agar (PDA) medium, insert five hyphae trays into SCM medium and incubate at 28 °C for 3 weeks.
Before sowing the wheat kernels, sprinkle 5 g of SCM medium in pre-washed sand soil and leave for two days, when sowing, the wheat kernels are externally sterilized with 5% Clorox ® for 5 min and rinsed several times with tap water followed by rinsing with deionized water.
According to the preliminary study results, the sterilized kernels were divided into three groups: the first group was immersed in distilled water, the second group was perfused with 5 mMCaCl2 solution, and the third group was perfused with 0.05 mM salicylic acid solution.
After 12 h, the soaking solution is removed and the kernels are washed with deionized water, each seed group is divided into two subgroups, one of which is sown in Fusarium graminis, one is pre-inoculated sand and the other is prewashed inoculated sand soil.
In a completely random design, the soil is enclosed in a plastic pot (35 cm in diameter × 27 cm deep) containing 8 kg of soil and treated six times.
In a completely random design of six treatments, the soil was confined to 35×27 cm plastic pots, each containing 8 kg of sand.
After seedlings, the pots are pruned into uniform 10 plants/pots, repeated 5 times for each treatment, and seedlings are collected after 21 days for morphological, biochemical, and molecular evaluation.
Osmotic protective molecules
When the evaluation was complete, we quantified the quaternary ammonium penetrant glycine betaine (GB) in the aqueous extract of dry wheat leaf powder.
The total free proline (TFP) content of wheat leaves was determined using KI-I2 reagents and GB-developed standards, followed by acid ninhydrin reagents and calibration curves created from proline.
Following the results of the study, total amino acids (TAA) in wheat leaf extracts were measured spectrophotometrically using glycine as a standard and ninhydrin-citrate buffer-glycerol reagent.
Fresh wheat leaves are then homogenized in liquid nitrogen and then extracted in phosphate buffer (50 mM, pH 7.0) to measure catalase (CAT) activity by monitoring the initial rate of H2O2 decomposition at 240 nm and calculating the activity using the extinction coefficient (40 mM-1 cm-1).
Increased light absorption at 560 nm due to photochemical reduction of nitroblue tetrazolium (NBT) to formazan, and the extinction coefficient (21.1 mM-1 cm-1) was utilized to evaluate superoxide dismutase (SOD) active leaf extract in wheat.
The color intensity due to the oxidation of hydrotriphenol to red gallin was monitored at 420 nm and polyphenol oxidase (PPO) activity was calculated using the extinction coefficient (26.4 mM-1 cm-1).
In the presence of phenylalanine as a substrate, the absorbance of the resulting transcinnamic acid at 290 nm is used to detect phenylalanine ammonia-lyase (PAL) activity.
According to Wang et al., PAL activity was calculated using the molar extinction coefficient of trans-cinnamic acid (10.24 mM-1 cm-1). （2006）。 The activity of all measured enzymes is expressed as μM.g-1 FMmin.
Following the experimental guidelines, we extracted total RNA from wheat seedlings and generated complementary DNA using reverse transcriptase (RT) using reverse transcription of a total volume of 20 μL of RNA using a thermal cycler.
The reaction process consists of a 60 min enzyme activation cycle at 40 °C, followed by a second enzyme inactivation cycle at 95 °C for 5 min.
After the above steps, we performed three more pooled studies containing primer pairs of the genes studied: catalase (CAT), glutathione reductase (GR), pathogenesis related 2 (PR2), pathogenesis related 4 (PR4), metallothionein-like protein 1 (MT), or plant chelin synthase (PCS) in each reaction, and retrieved data at the extension stage.
During amplification, the initial dissociation phase is at 95 °C for 10 min, followed by 40 cycles of 95 °C denaturation for 15 sec, 60 °C annealing for 30 sec, and 72 °C extension for 30 sec.
In order to exclude the presence of non-specific amplicons, the melting curve was obtained and the β-actin gene was used as a reference, and the relative expression of the studied gene was quantified and calculated using the method developed by Livak.
At the same time, the experimental results showed that inoculation with Fusarium graminearum or initiation with Ca or SA, as well as their joint interaction, significantly affected the growth characteristics of wheat seedlings, such as bud height, bud water content, leaf area, root depth and root water content.
Compared with the healthy uninfected control, inoculation with Fusarium graminearum significantly reduced the bud height, leaf area, root depth and relative root water content in the aboveground part of wheat seedlings, but had no significant effect on the aboveground water content.
Compared with the control treatment, seed initiation with CaCl2 increased aboveground height and leaf area without affecting aboveground water content or root depth, and slightly reduced root water content of wheat seedlings.
On the other hand, compared with control seedlings, SA-treated wheat seedlings had no change in bud height, bud water content or root depth, a slight decrease in root water content, and a slight improvement in leaf area.
Although the combination of fungal infection and priming treatment (Ca or SA) promoted bud and root growth, wheat seedlings infected with SA and Fusarium graminears showed more stress areas than infected seedlings.
Overall, in plants triggered by Ca or SA solutions, Fusarium graminus had a lower effect on wheat seedling growth than no infection, and the molecular impact was gradually reduced.