Silicon Root Irrigation Enhances Barley Resistance to Fusarium Head Blight

Nachaat Sakr

ORIGINAL ARTICLE

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Silicon Root Irrigation Enhances Barley Resistance to

Fusarium Head Blight

Nachaat Sakr

1 Department of Agriculture, Atomic Energy Commission of Syria (AECS), Damascus, P.O. Box 6091, Syria

*E-Mail: [email protected]

Received January 21, 2024

Silicon (Si) is recognized for its protective role in decreasing disease damage when absorbed by barley plants and has been proposed as a possible solution against Fusarium head blight, associated with devastating agronomic effects on overall yield and grain quality. However, root treatment of exogenous Si irrigating to enhance host resistance to Fusarium infection is unknown. For this purpose, a series of greenhouse experiments was conducted to examine the effects of Si irrigation at 1.7 mM to roots on pathogen development in barley heads. Two barley cultivars with contrasting FHB resistance (moderately resistant Arabi Aswad, AS, and moderately susceptible Arabi Abiad, AB) and infected with four Fusarium species with diverse pathogenicity were used. The quantification of the disease was through the determination of the disease incidence (DI, Type I resistance), disease severity (DS, Type II) and area under disease progress curve (AUDPC) calculated on the basis of DI and DS. Si absorption in barley enhanced the defense system in head tissues to pathogen invasion; FHB developed more severely on AS and AB plants grown without Si irrigation than on plants supplied with Si. Barley plants treated with exogenous Si irrigating were associated with a reduction of up to 19.3%, 19.8%, 18.7%, and 20.0%, respectively, in DI, DS and AUDPC calculated on the basis of DI and DS. Si contributed to the reduction of FHB in barley, especially for the moderately resistant cultivar; however, Si reduced the intensity of FHB in AB to a level comparable with AS. Importantly, Si treatment at 1.7 mM decreased disease damage FHB in previous bio-trials conducted on AS and AB under in vitro and field environments, showing that Si enhanced the expression of resistance to FHB infection in seedlings and adult barley plants. Taken together, the link of Si and host resistance provided a greater decrease in head blight in which both cultivars had augmented performances upon exogenous Si irrigating to roots; highlighting that Si is a potential safe and efficient policy to defend barley when invaded by Fusarium.

Key words: barley resistance, Fusarium species, Hordeum vulgare resistance, integrated disease management, silicon root irrigation.

Barley (Hordeum vulgare L.) is one of the oldest cultivated grain crops ranking fourth in importance, among small-grain cereals, and successfully cultivated under a board range of climates. From an economic point of view, its importance arises from being largely utilized for feed and food production worldwide. Almost 80%-90% of H. vulgare grain production is appropriated for livestock feed, while the remaining 10% is transformed into distilling, baking and malt for brewing (FAW, 2022). Its yield is directly menaced by diseases, such as Fusarium head blight (FHB), one of the most common and noxious fungal diseases of barley (Ogrodowicz et al., 2020). A pathogen complex of several Fusarium species can cause blight in head tissues (Fernando et al., 2021); the most globally pathogenic and distributed causal agents encompass F. graminearum and F. culmorum (Inbaia et al., 2023). FHB leads to premature brownish discolouration of florets scattered throughout the spike, which results in the formation of discoloured grains (brown, pink, red, orange or tan) (Dahl and Wilson, 2018). It results in considerable losses in crop production, quality and safety due to the risk of kernel contamination by a large variety of mycotoxins (Janssen et al., 2018).

Breeding resistant H. vulgare cultivars is an efficient policy to defeating FHB. Head blight resistance of barley (Sakr, 2022a) is a quantitative trait expressed from several quantitative resistance loci and linked with two types: resistance to floret infection (Type I) and spreading of the infection within the head (Type II) (Dahl and Wilson 2018; Janssen et al., 2018). However, commercial cultivars resistant to Fusarium development in head tissues are not yet obtainable to growers due to FHB resistance of H. vulgare is complex to use (Ogrodowicz et al., 2020). Furthermore, efficient chemical management of FHB resulting in potential environmental damage and significant economic losses is restricted to a small time window at around anthesis, when primary head invasion generally occurs (Janssen et al., 2018), and hence augmenting complexity of effective FHB control (Fernando et al., 2021). In novel years, the issue of FHB-barley has been further complicated by important interactions among

management techniques, pathogenicity of diverse Fusarium organisms and continual extreme climatic events (Sakr, 2022a). Hence, an alternative eco-friendly policy must be recognized in the near future to reduce damages due to FHB (Dahl and Wilson, 2018).

A progressively common policy is to increase the defense of plants against pathogens. In this scenario, the most promising approach is the use of silicon (Si) fertilization as part of an integrated disease management approach (Debona et al., 2017). In spite of Si has not been referred a major element for higher plants (Kaur and Greger, 2019), it has been shown to be helpful for the healthy development and growth of several plants exposed to biotic and abiotic constraints (Guo-Chao et al., 2018). The existence of soluble Si in plant shoots has been proven to decrease the intensity of several economically important fungal diseases (Deshmukh et al., 2016). The action mechanism of Si in host defense is linked with three actions, viz., the physical, biochemical, and molecular action of mechanisms (Debona et al., 2017), identified as formation of a silicon-cuticle double layer, cell wall stiffness reinforcement, callose deposition, papillae formation, promoting host biochemical defenses via accumulation of hydrogen peroxide at infection sites, accumulation of antimicrobial compounds (pathogenesis-related proteins, phytoalexins, phenylpropanoids, and flavonoids), enhanced activities of defense enzymes, gene expression induced by stressors in plants, and signal transduction (Guo-Chao et al., 2018; Kaur and Greger, 2019). In plants, the terminal Si level depends on various factors, encompassing host species, host age, the cultivar's ability to absorb and distribute Si around the plant, as well as the content of Si obtainable in soil solution (Deshmukh et al., 2016).

H. vulgare is recognized as a Si-absorber and absorb relatively high quantities of Si in shoots and leaves (Guo-Chao et al., 2018; Kaur and Greger, 2019). Fungal diseases of barley including powdery mildew (Blumeria graminis f. sp. hordei) and spot blotch (Bipolaris sorokiniana) have been reported to be decreased by Si application through a better performance of the primary metabolism of H. vulgare

plants (Wiese et al., 2005; Holz et al., 2022). In recent years, Sakr (2021b) reported encouraging findings with Si and FHB-barley; Si decreased the invasion rate and Fusarium spreading in barley head tissues by increasing Type I and Type II under controlled conditions. A little success was obtained with soil amendments than with foliar applications, showing that Si rates augmented in plants fed Si via the roots and leaves (Sakr, 2021b). Interestingly, root irrigating with Si has proven to reinforce the root growth resulting in its enhanced capacity of Si absorption and a better resistance to fungal infection in some pathosystems, i.e., Blumeria graminis f.sp. tritici causing wheat powdery mildew, Magnaporthe oryzaei causing rice blast, Rhizoctonia solani causing rice sheath blight, Phakopsora pachyrhizi causing Asian soybean rust (Zhang et al., 2003; Guevel et al., 2007; Arsenault-Labrecque et al., 2012; Du et al., 2022) including a decrease of FHB development on wheat (Sakr, 2022b; Sakr and Kurdali, 2023b). Thus, researches on whether exogenous Si irrigating to roots could have a positive function on H. vulgare resistance to Fusarium invasion in head tissues appears completely necessary but are still unknown.

Si advantage is crucial for sustainable production of barley (Sakr, 2021b), taken into account the worldwide dominance of Fusarium species (Ogrodowicz et al., 2020; Fernando et al., 2021). To our knowledge, this is the first report to explore the effect of Si irrigating via the root system on enhancing the components of barley resistance to FHB invasion under conditions controlling strictly all biotic and abiotic factors. Disease incidence (DI, Type I resistance), disease severity (DS, Type II) and Area Under Disease Progress Curve (AUDPC) calculated on the basis of DI and DS were evaluated in two barley cultivars with different FHB resistance levels supplied or not with Si. The ability of Si to enhance resistance on moderately susceptible to a level comparable to moderately resistant not amended with Si was also investigated. In addition, the relationships were examined between the current findings and the damage of FHB in previous analyses conducted on barley cultivars under in vitro and field environments to check whether Si could increase the expression of resistance

to FHB infection at the earliest and latest barley development stages during FHB infection.

MATERIALS AND METHODS

Barley materials and growth conditions

Two barley cultivars of Syrian origin: Arabi Aswad (AS, moderately resistant) and Arabi Abiad (AB, moderately susceptible), pre-selected according to different resistance to FHB as assessed in inoculation trials under in vitro, controlled and field conditions (Sakr 2023a), were used in the experiments. AS and AB were selected due to their identical anthesis and maturity dates, and board usage in the market (Ceccarelli et al., 1987). Prior to being utilized in trials, AS and AB seeds were surface sterilized by immersion in 0.5% sodium hypochlorite solution (NaOCl) for 1 minute. The seeds were then rinsed three times in sterile water. Eight surface-sterilized barely seeds were sown in plastic pots (20 x 15 cm) containing 2 kg of pasteurized soil. Using a gamma irradiator (ROBO, Russia), the pasteurization of the soil was carried out at 5 k Gy of Gamma Ray with Co60 source. The soil used was air dried and sieved (3 mm). The physicochemical characteristics of the clay soil (57% clay, 39% loam, 2% sand) used in the study were as follows: organic matter of 1.25%; K, Na, Ca, Mg = 1.81, 2.99, 33.1, 14 mg/100 g soil respectively; P = 13.4 mM and pH 7.8. After seedling emergence, they were thinned to five plants per pot, and nitrogen, in the form of urea, was applied at 0.173 g/pot at two dates: emergence and tillering.

Throughout the experiment, the plants were kept under controlled climatic conditions in a growth chamber with air conditioning for temperature control. Average daily temperature was set to 20°C day/night, 16 h of per day with a relative air humidity of 60%. Inoculation procedure

Till now, the recovery of head blight pathogens has not been achieved from Syria barley plants. Nevertheless, Fusarium species are frequently recovered form naturally infected wheat fields and known to strongly infect barley spikes (Sakr, 2023a). All Fusarium isolates, including six F. solani, five F. culmorum, f our F. verticillioides (synonym F. moniliforme) isolates and one F. equiseti isolate, were

isolated from naturally infected wheat grain. The 16 isolates were monosporic derived cultures of the original field isolates and were selected for their contrasting pathogenicity based on previous several experimental observations (Sakr, 2022a, 2023a). On Petri dishes with potato dextrose agar (PDA) with 13 mg/l kanamycin sulphate added after autoclaving, the isolates were morphologically identified with the aid of the Leslie and Summerell (2006) manual on the basis of microscopic studies of the shape and size of macro- and micro-conidia, and were molecularly distinguished by Random amplification of polymorphic DNA markers (Sakr, 2023a). These 16 Fusarium isolates were used in previous experiments conducted under in vitro, growth chamber and field trials to assess resistance levels of AS and AB (Sakr, 2022a, 2023a), and resistance levels were correctly and precisely distinguished.

Single spore isolates were stored short term on PDA at 4°C and long term by freezing at-16°C or in sterile distilled water at 4°C, and fresh cultures were produced on PDA medium. After 14 days, conidia were collected in sterile distilled water (SDW). Then, the suspensions were filtered through two layers of sterile cheesecloth to remove agar and adhering mycelia. The spore concentration was adjusted prior to use with the aid of a Neubauer chamber under an optical microscope and diluted to 5 x 104 conidia/ml as inoculum sources. Disease evaluation

The quantification of the disease resistance in AS and AB separately inoculated with the 16 Fusarium cultures was through the determination of the incidence of FHB (DI, Type I resistance), severity of FHB (DS, Type II) as well as area under disease progress curve (AUDPC) calculated on the basis of DI and DS. At full flowering (GS=65) and by using a conidial suspension for the 16 Fusarium isolates, barley plants were spraying inoculated on the whole head for bleaching of spikes (DI) or by inoculation in the central floret of the head (single flower inoculation) for bleaching of spikelets (DS). For the single spikelet inoculation method, 10 Ml of inoculum suspension was injected between the palea and lemma in the central floret of the head, utilizing a sterile micropipette tip. Each isolate was used independently (not mixed). Non-inoculated AS and AB

plants were sprayed with SDW and exposed to the same conditions as the inoculated plants. The both types of inoculations, head and floret, were made on the discrete heads of the same cultivar in two separate experiments. Regardless of inoculation method, immediately after inoculation, plants were maintained inside plastic bags the inner surfaces of which had been sprayed with SDW. AS and AB plants were kept under these conditions for 48 h in order to provide humid conditions favorable for the initial phase of pathogenesis.

DI, DS and AUDPC calculated on the basis of DI and DS were determined to decide the degree of Fusarium infection in light of visual damages in head tissues. DI was measured by counting the number of heads with obvious FHB symptoms (i.e., partially or fully bleached heads) and was expressed as the percentage of barley heads with symptoms. FHB severity was expressed as percentage of spikelets with clear FHB symptoms in comparing with the total number of spikelets per head as described in Sakr (2023a). AUDPC for each plant in each treatment and experiment was calculated on the basis of DI and DS using the trapezoid integration of the disease progress curve (Jeger et al., 2001) over the progressive blighting of heads scored at 7, 14, 21 and 28 days post inoculation. AUDPC was conducted with the beginning of heads with discoloured spikelets that are indicative of head blight about 1 week after inoculation.

Silicon application

Pure Si was employed to avoid the confounding impact of other nutritive elements in some Si-based soil amendments (Deshmukh et al., 2016). The silicon source was an analytically pure SO2 powder (H4SO4, Kieselsaure, Carl Roth GmbH + Co. KG, composed by a minimum silicon content of 99% Si), which was supplied along with the irrigation solution. In a previous study conducted on barley (Sakr 2021b), a SO2 powder decreased FHB damage in head tissues expressed by DI and DS of pathogenic Fusarium isolates following artificial spike and spikelet inoculation under controlled conditions (Sakr, 2021b), therefore, SO2 powder was preferred as Si source in the current research. Si as irrigation solution was prepared by dissolving a SiO2

powder in demineralized water. From the sowing till the fungal spraying time, the barley plants were weekly irrigated with 300 ml (per pot) with a concentration of 1.7 mM Si. The control was irrigated with 300 ml of SDW. AS and AB were watered with the similar volume of irrigation solution in the existence of Si (1.7 mM Si) or not.

Experimental design

The trials were carried out to correctly and precisely assess the influence of root treatment of exogenous Si irrigating into AS and AB on head blight DI, DS and AUDPC calculated on the basis of DI and DS (Figure 1). A 2 x 2 x 16 factorial experiment, consisting of two Si concentrations (0 and 1.7 mM, referred to as -Si and +Si plants thereafter), two barley cultivars with different resistance levels: AS and AB, and 16 Fusarium isolates causing FHB, were arranged in a randomized design with three replications. The experiment was repeated twice.

Si ability to enhance barley resistance under several experimental conditions

The bio-efficacy of Si to increase AS and AB barley resistance to FHB disease was measured by latent period (LP) of detached leaf inoculation, area under disease progress curve (AUDPC) of Petri-dish inoculation and coleoptile length reduction (CLR) of a coleoptile infection under in vitro conditions (Sakr, 2023b) (Table 1). The reduction in head blight symptoms due to the effect of Si was also expressed under field conditions as DI (Type I resistance), DS (Type II) and Fusarium-damaged kernels (Type III) (Sakr and Kurdali, 2023a) (Table 1). Therefore, we were able to examine the relationships between the current findings with the previous results of in vitro and field environments to check whether Si could increase the expression of resistance to FHB infection at the earliest and latest barley development stages during FHB infection.

Statistical analyses

Prior to ANOVA, the percentages of DI, DS and AUDPC calculated on the basis of DI and DS were arcsine angular transformed to stabilize variances. Data were subjected to factorial analysis of variance

(ANOVA) and the means of treatments compared by Fisher's least significant difference test at p<0.05 using the DSAASTAT add-in version 2011. Single degree-of-freedom contrasts test was used to make comparisons between AB supplied with Si and AS non-supplied with Si. Comparison among FHB isolates infected AS and AB upon Si treatment was made by the contrast procedure.

RESULTS

Si application reduced disease development of Fusarium pathogens in barley

ANOVA showed that Si, cultivar, cultivar x Si interaction had highly significant effects (p < 0.05) on DI, DS as well as AUDPC calculated on the basis of DI and DS (data not shown). On AS and AB, Si resulted in lower disease intensity based on the percentage of diseased florets and smaller necrotic patches, and less bleaching of the florets and discoloured kernels as compared to fungal-inoculated-controls (Figure 2). Regardless of botanical and pathogenic background for the host and fungal materials in the present research, FHB DI, DS and AUDPC calculated on the basis of DI and DS (Figure 3) were significantly decreased in +Si plants AS and AB barley plants inoculated with all analyzed Fusarium isolates relative to -Si plants, this indicates that root Si irrigation (1.7 mM) protected any tested barley cultivar whatever its quantitative resistance levels from infestation with any FHB pathogen whatever its pathogenic level under controlled environmental conditions. -Si barley plants supplied with SDW did no show diseased symptoms. In plants supplied with Si, DI, DS and AUDPC calculated on the basis of DI and DS were 19.3%, 19.8%, 18.7%, and 20.0%, respectively, smaller than in -Si barley plants. Si absorption in barley enhanced the defense system in head tissues to pathogen invasion; FHB developed more severely on AS and AB plants grown without Si irrigation than on plants supplied with Si. AS was significantly more resistant to head blight infection than AB (Figure 3).

Si (1.7 mM)-supplied cultivars, AS which is moderately resistant and AB which is moderately susceptible, had a DI that was lower by 23 and 14%, respectively in experiment 1; 22 and 19%, respectively in experiment 2, 20 and 18%, respectively in experiment 3 than plants without Si (Figure 3, A).

Figure 1. A schema of the experimental design in the study

Table 1: Influence of silicon at 1.7 mM in barley (Arabi Aswad, AS and Arabi Abiad, AB) resistance to Fusarium head blight measured by latent period (LP, days), area under disease progress curve (AUDPC) and coleoptile length reduction (CLR, %) under in vitro conditions, disease incidence (DI, Type I resistance), disease severity (DS, Type II resistance) and Fusarium--damaged kernels (FDK, Type III resistance) under field conditions

LP