Exploiting of pipazethate HCL as a plant activator targeting a salicylic acid pathway in rice for blast fungus Pyricularia oryzae resistance

doi:10.21203/rs.3.rs-4404973/v1

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Exploiting of pipazethate HCL as a plant activator targeting a salicylic acid pathway in rice for blast fungus Pyricularia oryzae resistance

https://doi.org/10.21203/rs.3.rs-4404973/v1

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Globally, blast disease in rice is one of the most important challenges limiting affected on rice crop productivity. Systemic acquired resistance (SAR) has been broadly investigated in a variety of plant species for enhancing inner resistance to various phytopathogens. The present study aimed to investigate the potential impact of pipazethate HCL as a substance of SAR against Pyricularia oryzae in rice. The obtained results showed that pipazethate HCL has no effectiveness of P. oryzae on liner growth with various concentrations in vitro. However, it significantly improved the level of resistance to rice blast fungus under greenhouse conditions with low and high concentrations under greenhouse conditions. Furthermore, pipazethate HCL, salicylic acid, and tricyclazole 75% are significantly decreasing the number of spores produced on infected rice lesions. Additionally, pipazethate HCL activates some salicylic acid (SA) signaling pathway indicators such as OsWRKY45 and OsNPR1, but jasmonic acid (JA) pathways such as AOS2, JAMYB, and PBZ1 (OsPR10) are not clear. The results suggested that pipazethate HCL is a salicylic acid analog against blast disease in rice inducing SAR that might be interrelated with the defense pathways of SA.

Pipazethate HCL

Pyricularia oryzae

Rice blast

Salicylic acid

SAR

Cultivation of Rice (Oryza sativa) spans approximately 160 million hectares of land (Kumar et al. 2019) and yields approximately 523.9 million tons of grain annually (FAOSTAT 2023), supporting with the significant amount like 3.5 billion individuals (Wing et al. 2018). In Egypt, production of rice has reached 4.84 million tons (FAOSTAT 2023). It has been suggested that rice production needs to increase by more than 40% by 2030 to meet growing demand (Khush 2005). However, plant diseases cause significant yield losses and pose a consistent threat to global food security (Pennisi 2010). Rice blast disease, caused by Pyricularia oryzae Cavara, poses a serious global challenge, leading to significant decreases in crop yield and quality (Agrios 2005). It is widely acknowledged as a major factor limiting rice yields worldwide (Asibi et al. 2019). The extent of yield losses varies depending on geographical regions and rice cultivars. In Japan, damages range from 20–100% (Khush and Jena 2009); in Indonesia, they range from 15–35%, and in the Philippines, from 50–85% (Suprapta et al. 2014). In Brazil, cultivar Colosso has experienced losses of up to 100% (Prabhu et al. 2009); in Korea, losses reached 14% (Shafaullah et al. 2011); in China, losses can be as high as 70% (Suprapta 2012). In Egypt, during epidemic seasons, the impact may range from 30–50%, while in normal or mildly affected seasons, susceptible cultivars can experience an annual production decrease of approximately 5% (Sehly et al. 2002). The susceptibility of cultivars, environmental factors, and disease management practices significantly influence the occurrence and severity of this disease (Chung et al. 2022).

Strategies for managing rice blast disease typically involve recommending resistant cultivars, utilizing fungicides, and exploring biotechnological approaches (Sahu et al. 2022). However, developing resistant cultivars can be challenging due to the susceptibility of the rice genome to molecular genetic manipulation and mutations (Jeon et al. 2007). Additionally, the use of fungicides is becoming less favorable due to their adverse effects on the environment and human health (Coca et al. 2006). Moreover, the repeated application of fungicides can lead to the emergence of new physiological races of P. oryzae that exhibit resistance to fungicides in natural settings (Sawada et al., 2004; Suzuki et al. 2010).

Moreover, systemic acquired resistance (SAR) stands out as a distinctive signal transduction pathway crucial for enhancing plant resistance against invading pathogenic organisms (Gao et al. 2015). This pathway operates by elevating levels of salicylic acid (SA) and inducing the accumulation of pathogenesis-related proteins (PRs) (Grant and Lamb 2006). Notably, SA and benzothiadiazole (BTH) enhance basal defense responses in both monocots and dicots (Stadnik and Buchenauer, 1999). Synthetic inducers, such as BTH, have been observed to increase resistance in rice against Rhizoctonia solani (Rohilla et al. 2002) and Magnaporthe oryzae (Sugano et al. 2010).

Additionally, salicylic acid (SA), jasmonic acid (JA), azelaic acid, and DL-β-aminobutyric acid (BABA) have emerged as popular agents for studying defense mechanism of rice (Conrath 2011). On the other hand, Pipazethate HCL, a compound known chemically as 2-(2-piperidinoethoxy) ethyl 10H-pyrido [3,2-b] [1,4] benzothiadiazine-10-carboxylate hydrochloride, functions as a non-narcotic oral antitussive substance (El-Saharty et al. 2010) by acting centrally on the medullary brain cough center (Vakil et al. 1966). The objectives of the current study were to (i) screening pipazethate HCL effect inducing resistance to P. oryzae compared with salicylic acid and the fungicide tricyclazole 75% in rice, and (ii) explore the potential pipazethate HCL resistance mechanisms related to the defense pathways of SA and JA.

Plant materials and growth conditions

Egyptian rice cultivar seeds (Sakha 101), which is susceptible to blast disease, were obtained from the Rice Research and Training Center (RRTC), Agricultural Research Station (ARC), Sakha, Kafr El-Sheikh, Egypt. The obtained seeds were surface sterilized with 1% NaClO (sodium hypochlorite) for 2 mins and then rinsed with sterile water. Pots (15 cm diam.) were used for seeds sowing and filled with sterilized soil under greenhouse conditions including controlled temperature (28^oC), relative humidity (80%), and light/dark period (12/12h). Three-week-old seedlings with contained 3–4 leaves were used for the inoculation using P. oryzae spore suspension.

Isolation of Pyricularia oryzae isolate

This study was carried out in Rice Pathology Research Department, Plant Pathology Research Institute, ARC, Sakha, Kafr El-Sheikh, Egypt. Typical blast-infected tissues from Sakha101 rice leaves cultivar samples were collected from Kafr El-Sheikh governorate over the growing season in 2016, samples were prepared by cutting into pieces (1-2-cm). The samples underwent a surface sterilization process by being immersed in a 0.5% NaClO (sodium hypochlorite) for 2 mins and then rinsed thrice with sterile water. After sterilization, the pieces were placed on sterile filter paper in 9-cm-diameter Petri dishes and incubated at 25°C for 24 h under continuous fluorescent light. Following this, a solitary conidium was produced by streaking conidia onto water agar medium (WA) within 24 h from sporulating lesions. Subsequently, single conidium was selected to transfer into WA for an additional 24 hours. The hyphal tip was identified using a compound microscope and transferred to banana dextrose agar (BDA) (200 g banana + 15 g dextrose + 20 g agar + 1 L distilled water). Pure culture of P. oryzae was maintained for further studies.

Race identification and resistance genes

Race identification of P. oryzae isolate was conducted using 8 different varieties called international differential varieties (IDVs) i.e. (Raminad str, Zenith, NP-125, Usen, Dular, Kanto 51, CI 8970 s and Caloro) (Atkins 1967). Moreover, ten international Japanese differential varieties (IJDVs) i.e. (Shin 2 (Pi-K^s), Toride 1(Pi-Z^t), Tusyake (Pi-K^m), Kanto 51(Pi-K), Fukunishiki (Pi-Z), Ishikarishiroke (Pi-i-Pi- K^s), BL-1(Pi-b), Yashiro-ochi (Pi-ta), Pi No.4 (Pi-ta²), and Ashia Asahi (Pi-a) were used to determine the effective gene for the blast resistance. These trials were carried out with artificial inoculation under greenhouse conditions.

Inoculum preparation of P. oryzae

Purification and identification for P. oryzae isolate which was isolated from Sakha101 cultivar were established on medium of BDA. Pure isolate was cultured at 28°C in the absence of light on BDA. After week, the plates were flattened and exposed using fluorescent light to stimulate sporulation for 3 days at 28°C and then conidia were collected following the protocol (Thuan et al., 2006). The concentration of the inoculum was adjusted to 5 x 10⁴ spores/ml using hemysytometer. Then 0.5% gelatin was added to improve the adhesion of spores to leaf surfaces (Bastiaans 1993).

Tested Chemical materials

Two chemical substances (Pipazethate HCL and Salicylic acid) in comparison with the recommended fungicide (tricyclazole 75%) (Fig. 1) were prepared to assess their efficiency in disease management. Pipazethate HCL was obtained from Egyptian International Pharmaceutical Industries Company (EIPICO), the stock solution was directly suspended in de-ionized water with eight different pipazethate HCL concentrations (10, 25, 50, 125, 250, 500, 1000 and 2000 mg/L.). Moreover, fresh suspensions were continuously mixed to prevent sedimentation before application.

Salicylic acid (powder contains 99.99% active ingredients) was obtained from Sigma Aldrich‎ dissolved with few drops of 70% ethanol. It was applied at the rate of 1000 mg/L of concentration for the experiments. Moreover, the treatment of fungicide was prepared in the form of a water-dispersible granule contained 75% active ingredients of tricyclazole. This fungicide was mixed in sterile water and applied with the rates of 0.5 g/L. Five days before inoculating the plants with the pathogen, these substances, coupled with water containing a small amount of ethanol for control purposes, were applied.

In vitro assay of P. oryzae

Application of pipazethate HCL at different concentrations (0, 10, 25, 50, 125, 250, 500, 1000 and 2000 mg/L.) and salicylic acid (1000 mg/L) were tested for their effect on the radial growth of P. oryzae. A sterile cork borer was used in order to make five wells on BDA plates for individually concentration. A 5 mm disks of P. oryzae isolate were putted in the center of each BDA 9 cm petri dish. The other four wells were filled individually with four concentrations of the tested chemical substance. Plates were kept in an incubator at 28°C, and the fungal growth was assessed every 48 hours until the control plates exhibited complete growth. Also, the growth inhibition was defined as the percentage inhibition rate of radial growth relative to the control. (Ghildiyal and Pandey, 2008).

(Inhibition percentage of radial growth= (R₁- R₂ / R₁) ×100) where:

R₁ control value represents the largest distance grown by the test fungus in the direction of the maximum radius. R₂ represents the distance between the inoculum of fungus and the treatment

Disease assessment under greenhouse conditions

A design of complete randomization (CRD) comprising five replications. (20 plants per replicate) was used to assess the efficiency of the tested chemicals against rice blast race ID-11under greenhouse conditions.

The spore suspension of P. oryzae was diluted to a concentration of 4 x 10⁵ spores/ml and applied using an electric spray gun. Control treatments included pots containing only the pathogen and pots inoculated solely with the chemical substance, aimed at assessing any potential side effects on plants. The infected pots were placed in a dark chamber with a humidity level of 90–95% and a temperature of 25–28°C overnight, before being transferred to greenhouse conditions with periodic sprinkling to promote infection development.

Disease severity was evaluated 7 to 10 days after inoculation using the standard 0–9 scale (Cai et al., 2008) (Table 1) according to the following equation (IRRI 2013).

Table 1

Disease assessment scale used in the current study
Scale	Description	Diseased leaf area (%)
0	No typical susceptible lesion observed	0
1	Rapid observation does not reveal leaf lesions, but careful scrutiny of each row reveals few lesions.	< 0.3
2	Rapid detection detects a few lesions.	0.3–0.9
3	Several lesions are randomly scattered within a plot, and the lesion number on an infected leaf ranges from 1 to 4.	1–2
4	Upper leaves are uniformly dotted with blast lesions but without necrotic (brown) leaf tips. A few to several leaves are brown.	3–7
5	Several to many lower leaves become necrotic and few dead leaves are observed. Tips of several upper leaves show brown color and begin to fold.	8–14
6	Lower leaves are uniformly exhibiting brown color and several dead leaves are visible. Tip necrosis of upper leaves is predominant.	15–24
7	Tips of most upper leaves are curling. Middle and lower leaves are brown. Several plants or tillers are stunted or dead	25–39
8	Extensive leaf curling and browning of upper and middle leaves are prevalent. Plants re generally stunted and many plants are dead.	40–65
9	Majority of plants are severely stunted, brown and dead. Only few to several plants have green leaves with heavy infection.	> 65

$$(Disease Leaf severity=\sum \left[\frac{r х {n}_{r}}{9 х {N}_{r}}\right] х 100)$$

Where r is the rating value, n_r is the number of infected leaves with a rating of r and N_r is the total number of tested leaves.

Blast lesion spore production and Chlorophyll content

Four blast lesions from three replicates were transferred into a 1 ml test tube containing sterile water, and vigorous shaking was performed to dislodge the spores. Subsequently, the spore suspension was examined using hemocytometer in order to calculate number of spores per lesion. Spores number was counted according to the following equation.

$$(Number of spores per lesion=\frac{Number of spores \text{х}2000 \text{х} Quantity of water taken}{ Number of lesions} )$$

To assess chlorophyll content one week after inoculation, the chlorophyll meter (Mod. Spad-502, Minolta Camera Co. Ltd., Japan) was employed to measure the chlorophyll levels in rice leaves.

Leaf samples and RNA extraction

Leaf samples were collected freshly after the treatments (12, 24, 36, 48, 72, and 96h). The inoculation was administered 24 hrs of post-treatment. with three replications for each treatment. Total RNA was extracted by Trizol reagent kit in accordance with the protocol provided by the manufacturer's instructions (iNtRON Biotechnology, Inc.).

The quality of RNA integrity was confirmed through 2% agarose gel electrophoresis stained with ethidium bromide. A consistent RNA concentration of 2 µg was employed for cDNA synthesis, utilizing the Intron-Power cDNA synthesis kit (Cat. no. 25011).

Analysis of gene expression.

The levels expression of some resistant -related genes, including specific primer OsWRKY45 (Os05g0322900), OsNPR1(Os01g0194300), JAMYB (Os11g0684000), OsAOS2(Os03g12500), PBZ1 (OsPR10) (Os12g36880), and actin (Os03g0718100) as a housekeeping gene for the gene expression normalization. Total reaction mix of 20 µL was used, contained 10 µl of SensiFast™ SYBR Lo-Rox master mix (Bioline, United Kingdom), 0.5 µM of each primer, 2 µl of cDNA and 7 µl RNase- free water. Tests were completed in duplicates using the Stratagene MX300 P real-time PCR system (Agilent Technologies). The reaction, thermal cycles, and primer sequences employed for the RT-PCR analysis were conducted following the procedures outlined in our previous study. The relative levels of gene expression were assessed using 2 − ΔΔct method Livak and Schmittgen (2001). The fold change for each gene was adjusted with respect to (actin) and aligned with the standards established by the control plants.

Statistical analysis

Throughout this study, Data were subjected to analysis of variance (ANOVA) using CoStat Computer Program version 6.311. Statistical comparisons of means were conducted using multiple range tests, as per Duncan's method Duncan (1955). Moreover, the schematic workflow of the current study was presented in Fig. (2).

Race identification and resistance genes

Three cultivars of the international differential rice varieties (Usen, Kanto 51 and Caloro) revealed susceptibility to P. oryzae isolate, whereas five cultivars (Raminad str, Zenith, NP-125, Dular and CI 8970s) demonstrated resistance to rice blast. According to the IDVs, the tested isolate was confirmed as P. oryzae race ID-11 (Table 2). The reactions of rice Japanese varieties to artificial infection of rice blast disease at Rice Research and Training center were tested under greenhouse in order to determine the effectiveness of resistance genes (R-genes). Data in Table (2) revealed that two R-genes (Pi-K and Pi-ta²) were breakdown and eight R-genes (Pi-K,^s Pi-Z,^t Pi-K^m, Pi-Z, Pi-i-Pi- K,^s Pi-b, Pi-ta and Pi-a ^s) were resistant to P. oryzae fungus.

Table 2

Reaction of the international differential varieties and Japanese varieties to rice blast fungus isolate ID-11 under greenhouse conditions.
No.	IJVs			IDVs
No.	Genotype	R-gene	Reaction	Genotype	Reaction
1	Shin 2	Pi-K^s^s	R	Raminad str	R
2	Toride 1	Pi-Z^t	R	Zenith	R
3	Tsuyuake	Pi-K^m	R	NP-125	R
4	Kanto 51	Pi-K	S	Usen	S
5	Fukunishiki	Pi-Z	R	Dular	R
6	Ishikarishiroke	Pi-i-Pi- K^s	R	Kanto 51	S
7	BL-1	Pi-b	R	CI 8970 s	R
8	Yashiro-mochi	Pi-ta	R	Caloro	S
9	Pi No.4	Pi-ta²	S	-	-
10	Aichi Asahi	Pi-a	R	-	-
IJVs = International Japanese varieties
IDVs = International differential varieties

Effect of pipazethate HCL on the linear growth of P. oryzae in vitro

The antifungal activity of pipazethate HCL on P. oryzae was evaluated at various concentrations on petri dishes under laboratory conditions. Laboratory tests indicated that pipazethate HCL does not exhibit antifungal activity against the mycelial linear growth of the isolate P. oryzae (Fig. 3). The observed protective effect is not attributed to the antimicrobial activity of pipazethate HCL, but rather to the triggering of plant defense responses.

Pipazethate HCL- P. oryzae interaction in vivo

Although, pipazethate HCL treatments obviously reduced blast lesions on rice leaves (Fig. 4A), however, a slight toxicity on rice leaves as a blanching on the leaf tips with 2–3 mm (data not presented) was observed. The obtained results revealed that disease severity was 2.1, 12.76, 6.6, 16.19, 7.1, 33.64, 35.5, 28.9, 82.8% at concentrations 2000, 1000, 500, 250, 125, 50, 25, 10, 0 mg/L, respectively. The concentration of 125 mg/L had the best protective effect in all subsequent experiments.

On the other hand, all treatments significantly improved plant resistance against rice blast disease, however, there were considerable differences among all tested chemicals compared to control. Tricyclazole was the effective substance at concentration 500 mg/L with disease severity 1.62%, followed by pipazethate HCL and salicylic acid with 8.0 and 35.8%, respectively (Fig. 4B). Moreover, all treatments positively reduced spore production on rice blast lesion where tricyclazole75% recorded the lowest number of spores (262.5) followed by pipazethate HCL (1475) and salicylic acid with (1662.5), compared to control plants (Fig. 4C). Moreover, pipazethate HCL application led to protect rice plants against P. oryzae as a whole-plant assay compared to non-treated plants (control) (Fig. 5A and B). In addition, the spore’s density of rice blast pathogen was dramatically reduced by application of pipazethate HCL compared to control in the successfully developed lesions (Fig. 5C).

Chlorophyll content

Applications of the previous treatments led to a significant increase in chlorophyll content where there were significant differences in tricyclazole, pipazethate HCL, salicylic acid compared to control. The best result was recorded with tricyclazole (Fig. 6).

RT-PCR and gene expression analysis

The analysis of gene expression appeared an increase in salicylic acid (SA) levels, along with the corresponding transcripts, was observed following the treatment of pipazethate HCL and SA (Fig. 7). For instance, the application of both SA and pipazethate HCL treatments to rice leaves conferred resistance against rice blast fungus. HCL application was the trigger of resistance to blast fungus where pipazethate HCL may be considered as an endogenous promoter for induced resistance in the rice leaves. Salicylic acid and pipazethate HCL solutions were applied on rice leaves 24 h before P. oryzae inoculation resulting in decrease the investigated blast lesions number. Then, the results suggested that pipazethate HCL is a new plant activator against blast disease in rice, possibly because of the mechanism of salicylic acid signaling. In a qRT-PCR analysis of rice-P. oryzae interaction, the SA treatment significantly up-regulated the expression of the SA-responsive genes OsWRKY45 and OsNPR1. These results suggested that the treatment of salicylic acid activates rapid responses expression in rice leaves largely in SA signaling pathway (Fig. 7A and B).

SA signaling pathway

Expression level of OsNPR1 was found to gradually increase afterward 12 h of treatment by salicylic acid and reached to 3.2 fold after 24 h, also, Significant increases were observed 72 hours after inoculation, reaching up to 13-fold. Concerning the pipazethate HCL treatment and after 24 hours, there was a modest increase in gene expression, but substantial increases were observed 12 hours after infection, reaching up to a 24.3-fold change (Fig. 7A).

OsWRKY45 on SA pathway revealed dramatically increase after 12h of SA treatment where the fold change reach to 1.8-fold change, and reached to maximum expression after 24h of inoculation where recorded 9.6-fold change. At 12 hours, pipazethate HCL exhibited a lower increase in gene expression, with a 3.45-fold change., whereas at 24 h after inoculation large increases occurred up to 5.1-fold change (Fig. 7B), this result indicates the correlation between the gene expression influenced by pipazethate HCL treatment and the occurrence of blast infection.

JA Signaling pathway

Gene expression showed minimal increase 36 hours after SA treatment, but exhibited a significant rise 12 hours after inoculation. This observation suggests that while transcription factors such as JAMYB, PBZ1 (OsPR10), and AOS2 were not affected wiwth the treatment of SA, influenced their gene expression by infection with the blast pathogen, resulting in fold changes of up to 13.3, 14.5, and 14.5 respectively. This impact was consistent across all genes associated with JA signaling (Fig. 6C, D, and E). Conversely, the expression of gene following pipazethate HCL treatment showed an increase of up to 2.0, 2.2, and 2.3 folds after 12 hours of inoculation, followed by a gradual decrease until 48 hours after inoculation for JAMYB transcription factor, PBZ1 (PR10), and AOS2 genes, respectively (Fig. 7C, D, and E).

Systemic acquired resistance (SAR) is a phenomenon by which plants display increased of resistance levels to a wide variety of pathogens in response to the treatment of treatment by chemicals (Gao et al. 2015). Whereas, SA- and JA/ET-Related defense signals, synergistically or antagonistically, communicate with each other (Durrant and Dong 2004). However, chemical activators protect rice against diseases by initiating on defense signaling pathway of SA (Iwai et al. 2007; Shimono et al. 2007). In this study, our focus has been on the utilization of pipazethate HCL as a novel systemic acquired resistance (SAR) substance activator to enhance resistance against blast disease in controlled conditions. The obtained results explored that pipazethate HCL targets some SA pathway indicators, but JA pathways were not clear. Interestingly, this study represents the inaugural investigation into the agricultural application of pipazethate HCL as a substance activating systemic acquired resistance (SAR) in plant species.

In controlled experiments, applying pipazethate HCL foliarly at both higher and lower concentrations displayed clear benefits to improve the resistance to rice blast disease. This positive effect of salicylic acid and pipazethate HCL is probably linked to their ability to enhance salicylic acid (SA) signaling in leaves rather than jasmonic acid (JA) signaling. The strong relationship observed within gene expression in the signaling pathways of SA and JA, along with disease resistance in rice, underscores the importance application of salicylic acid.

Moreover, the expression of genes involved in the salicylic acid signaling pathway, particularly OsWRKY45, was notably pronounced at 12 hours after pathogen infection, whereas OsNPR1 gene expression was generally moderate but increased specifically at the 12-hour mark post-infection. This observation aligns with the recognized role of the SA pathway in enhancing resistance in rice (Jiang et al. 2009). Previous research has highlighted the importance of functional application of SA analogs such as INA, BTH, and probenazole, which induce pathogenesis-related (PR) genes expression conferring resistance against various plant pathogens. While the SA pathway triggers defense responses in plants through chemical signals, it does not directly affect plant pathogens. Pipazethate HCL can be regarded as an SA analog, as its application, similar to exogenous SA or SA analogs, activates resistance mechanisms against invading pathogens (Gao et al. 2015). These findings are consistent with previous studies that demonstrated the efficacy of SA application against wheat powdery mildew (Blumeria graminis f. sp. Tritici), bacterial blight of rice (Xanthomonas oryzae pv. oryzae), rice blast (P. oryzae), and rice sheath blight (Rhizoctonia solani) (Li et al. 2020; Liang et al. 2022; Sood et al. 2013; Qiu et al. 2007).

The gene expression of OsNPR1 and OsWRKY45 that related to the pathway of SA in pipazethate HCL application strongly up-regulated after 12 h of infection. This could elucidate the incorporation of this substance as an efficient stimulator of the salicylic acid pathway, aligning with the priming effect of pipazethate HCL. The application by BTH substance with high-doses that led to constitutive defense responses including various direct defense related genes ( Wise et al. 2016; Van Hulten et al. 2006). In rice, the SA path is divided into two paths that depend on co-transcription factors OsNPR1 and OsWRKY45 (Goto et al. 2015; Nakayama et al. 2013; Sugano et al. 2010 ). Additionally, WRKY45 plays an important role as a master regulator of the transcriptions in one of two branches in rice of the SA pathway in the resistance to BTH-induced diseases (Nakayama et al. 2013). OsWRKY45 serves as a crucial transcription factor in rice with the salicylic acid signaling pathway, playing a role in mediating chemical-induced resistance against various pathogens. Its constitutive overexpression imparts a high level of resistance against M. oryzae and (Xoo). (Goto et al. 2016). Moreover, It has been documented that Sakha 101 possesses the Pit, Pib, and Piz-t genes Hassan (2013), with WRKY45 establishing connections with R proteins such as Pi36, Pit, Pib, Pi-ta, and Piz-t. Consequently, WRKY45 may function as a central player in rice effector-triggered immunity (ETI). (Liu et al. 2016), which supports resistance by activating OsWRKY45.

Previous studies have shown that suppressing OsNPR1 in rice impedes BTH-induced resistance to M. oryzae by disrupting the salicylic acid signaling pathway (Feng et al., 2011; Shimono et al. 2007). Moreover, the gene expression of OsNPR1 increased resistance to rice blast disease upon application of pipazethate HCL, particularly evident at 12 hours after inoculation. Additionally, several studies have indicated that OsNPR1 is indirectly associated through NPR3, NPR4 (Fu et al. 2012), OsWRKY03 of SA pathway (Liu et al. 2005), OsWRKY71 (Liu et al. 2007), and PR10 (Chern et al. 2005). In Arabidopsis, NPR1 is the sole gene affecting the downstream direction of systemic acquired resistance (SAR) signaling in the SA pathway. NPR1 is also involved in the jasmonate- and ethylene-regulated, salicylic acid-independent (ISR) pathway (Ramirez et al. 2010).

In this current study, the investigation into the jasmonic acid (JA) pathway focused on three genes: JAMYB, AOS2, and PR10. The expression of Rice JAMYB is not influenced by salicylic acid but is stimulated by jasmonic acid, making it a significant inducer of systemic acquired resistance (SAR) in rice (Cao et al. 2015).

The JAmyb gene expression increased following the infection with P. oryzae both resistant and susceptible interactions, with a notably higher level observed in susceptible interactions. Furthermore, OsAOS2 played a crucial role in JA production during rice blast resistance and the activation of PR genes (Filipe et al. 2018). The expression of OsAOS2 in rice leaves can be significantly induced following infection with M. grisea (Mei et al., 2006). Additionally, Rice Protein PR10, originally identified as a probenazole-inducible protein and termed PBZ1 protein (Jwa et al. 2001), is induced by various substances such as SA and JA, as well as by microorganisms like Xoo and M. grisea (Huang et al. 2016; Kim et al. 2003, 2004).

Regarding the efficacy of pipazethate HCL in vitro, it showed no impact on the mycelial growth of rice blast fungus, suggesting that pipazethate HCL lacks antifungal properties. This observation is consistent with certain prior studies wherein analogs of salicylic acid (SA) and benzothiadiazole (BTH), functioning as synthetic chemicals, were found to induce resistance to rice blast disease in vivo without directly exhibiting antifungal effects (Cole 1999; Yoshida et al. 1990). These chemicals demonstrated an indirect effect against P. oryzae when applied foliarly by reducing infected lesions. Ogawa et al. (2011) noted that isotianil lacks antimicrobial activity in vitro but effectively induces immune responses against rice blast fungus. Additionally, induced resistance against rice blast using the culture filtrate of Bacillus subtilis DL76 was reported to reduce appressorium formation, conidia production, fungal growth, and pathogenicity (Kgosi et al. 2022).

Pipazethate HCL exhibited superior resistance against rice blast compared to the applied fungicide (tricyclazole 75%). However, further investigations are necessary to optimize the application of pipazethate HCL across various rice genotypes and plant species, considering diverse environmental conditions, including greenhouse and open-field trials. In contrast, the use of fungicides carries potential risks to food production, as well as associated with the effects on environment and human health. While some studies suggest the safety of tricyclazole, others have pointed out its adverse effects. These may include potential harm such as impairment of testosterone secretion and testicular structure, leading to negative effects on the sperm production system (Fattahi et al. 2015). Moreover, toxicological studies have demonstrated the safety of pipazethate HCL in animals, with an LD50 of 214 mg/kg in mice and 560 mg/kg in rats when administered orally (Prime, 1961). Hence, the use of eco-friendly and safe treatments such as pipazethate HCL as systemic acquired resistance inducers is recommended.

In conclusion, it is strongly recommended that additional comprehensive studies be conducted to confirm and establish the applicability and efficacy of pipazethate HCL as a novel activator within plant defense pathways for effective plant protection.

Rice blast disease is a devastating agricultural affliction, causing significant yield losses worldwide. Our study underscores the promising role of pipazethate HCL in inducing resistance against the blast disease of rice, with potential ties to the activation of the salicylic acid signaling pathway over the jasmonic acid pathway. Moving forward, it is imperative that future research endeavors delve deeper into exploring the application of pipazethate HCL across diverse environmental conditions and genetic backgrounds to fully assess its efficacy in safeguarding against this detrimental disease.

Acknowledgments: The authors express their gratitude to Prof. Dr. Khaled Abdelaal, Director of PPB Lab, and all members of PPB Lab and EPCRS Excellence Centre (Accredited according to ISO/17025, 9001, 14001, and OHSAS/18001), Department of Agricultural Botany, Faculty of Agriculture, Kafrelsheikh University, Kafr-Elsheikh, Egypt. Additionally, the authors acknowledge the Rice Pathology Department, Plant Pathology Research Institute, Agricultural Research Center, Kafr-El-Sheikh, Egypt.

Authors' contributions

EKS and RAA, AAE designed the research idea and methodology; EKS and RAA were involved in writing the manuscript; KEG, ESB and AAE provide materials and resources; EKS and ESB were involved in data collection; EKS and KEG were involved in data analysis; RAA, AAE and KD were involved in the reviewing and editing the manuscript. All authors read and approved the final manuscript

Funding: This study was supported by Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt and Faculty of Agriculture, Kafrelsheikh University, Kafrelsheikh, Egypt.

Data availability

All data and materials are available if requested.

Conflict of interest: The authors declare that they have no conflict of interest.

Ethical approval: This article does not contain any studies with human participants or animals performed by any authors.

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Exploiting of pipazethate HCL as a plant activator targeting a salicylic acid pathway in rice for blast fungus Pyricularia oryzae resistance

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Abstract

Figures

Introduction

Material and methods

Plant materials and growth conditions

Race identification and resistance genes

Tested Chemical materials

Disease assessment under greenhouse conditions

Blast lesion spore production and Chlorophyll content

Leaf samples and RNA extraction

Statistical analysis

Results

Race identification and resistance genes

Chlorophyll content

RT-PCR and gene expression analysis

SA signaling pathway

JA Signaling pathway

Discussion

Conclusion

Declarations

References

Additional Declarations

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