Evaluation of Medicinal Plant Extracts for Rice Blast Disease Control

Tan Yanping, Deng Shiqi, Qin Yonghua, Xu Xin, Yu You, Cui Liu, Wang Chuntai, Jiang Changjie, Liu Xinqiong

Letter

Evaluation of Medicinal Plant Extracts for Rice Blast Disease Control

Tan Yanping1, #, Deng Shiqi1, #, Qin Yonghua1, Xu Xin1, Yu You1, Cui Liu1, Wang Chuntai1, Jiang Changjie2, 3, Liu Xinqiong1

(Hubei Province Key Laboratory for Protection and Application of Special Plant Germplasm in Wuling Area of China; Key Laboratory of State Ethnic Affairs Commission for Biological Technology, College of Life Science, South-Central Minzu University, Wuhan , China; Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), Tsukuba 305-8602, Japan;)

The potential of medicinal plant extracts to control rice blast funguswas investigated. We screened 48 extracts prepared from 8 medicinal plant species and identified 20 extracts with ≥10% inhibitory activity against. Among them, ethanol extract of, ethyl acetate extract of, and-butanol extractofplants exhibited 100%, 70% and 50% inhibition ofspore germination, respectively. In contrast, extracts from,,,andshowed ≤ 30% inhibitory activity. Analysis using ethanol extracts prepared from different parts of.plants showed that the extracts of pericarps and roots had the highest inhibitory activity againstspore germination, with inhibition rates of 77% and 78%, respectively. Furthermore, spray treatment of rice seedlings with.pericarp extract significantly enhanced blast resistance, indicating that the extract can serve as a potent antifungal agent. The expression levels of rice defense genes,andwere significantly upregulated in response to pericarp extract application, suggesting that the pericarp extract also activates the host defense system of rice plants. These findings lay the foundation for both the development of plant-derived biopesticides for plant disease management and effective utilization of., an endangered medicinal plant.

Rice blast, caused by, is a major disease of rice plants (Yang et al, 2021). Each year, rice blast is responsible for 10%–30% of riceproduction losses, or up to half of global rice yield ($1 billion USD) (Han et al, 2019; Kim et al, 2020). Therefore, control of rice blast has been a primary focus in rice breeding. Currently, breeding and promotion of resistant rice varieties are considered as the most economical and effective approaches to control rice blast. However,has a wide variety of pathogenic races (Deng et al, 2017; Xie et al, 2019). Since the 1960s, with the promotion and cultivation of resistant rice varieties, pathogenic races ofhave increased in pathogenicity and genetic complexity, resulting in a marked decrease in resistance to this pathogen in rice (Yin et al, 2019). Chemical pesticides have been extensively used to control plant diseases (Liu et al, 2018), but their usefulness is becoming limited due to the emergence of chemical-resistant isolates, environmental pollution, changes in soil structure and composition, and health concerns (Zhai et al, 2019). Therefore, the research and development of novel biopesticides are more important than ever for sustainable agriculture (Baker et al, 2020; Tariq et al, 2020). Recent studies have shown that some plant extracts have potent antifungal effects, which may be used as natural plant protection agents. For rice blast disease, plant extracts fromL. (Abbruscato et al, 2014),(Ngo et al, 2019) and(Bae et al, 2021) have been shown to have antifungal activity againstand, and several antifungal components including saponins and terpenoids have been identified from these plant extracts.

Medicinal plants are rich in active metabolites with therapeutic effects and have been utilized to treat a variety of human diseases (Singh, 2015; Mehta and Dhapte, 2016).is a perennial medicinal plant in the liliaceae family. The roots of Rhizoma Paridis have great medicinal value, and these plants are widely distributed in the Wuling Mountains located in central China (Hubei, Hunan, Sichuan and Guizhou provinces) (Sharma et al, 2015; Chen et al, 2019; Wang et al, 2019). Rhizoma Paridis has been shown to have powerful pharmacological activities, including hemostasis, expectorant, analgesic, detoxification, anti-cancer (e.g. cervical cancer), anti-asthma and anti-microbial effects (Zhao et al, 2010; Shah et al, 2012; Duan et al, 2018; Puwein et al, 2018; Jin et al, 2021). Several steroidal saponins have been identified in Rhizoma Paridis, which account for the majority of bioactive compounds (Qin et al, 2012; Sharma et al, 2015; Liu et al, 2022). Meanwhile,is considered one of the 50 fundamental herbs in Chinese herbalism and has been widely used in the clinical intervention fordiabetic osteoporosis in Asia (Tian et al, 2020); andis used for treating tonsillitis, rheumatic arthralgia, diarrhea, dysentery, gastroenteritis, cardiovascular and thrombosis disorders (Hina et al, 2020). In addition,(Grover and Yadav, 2004),(Dong et al, 2018),(Wang et al, 2021),(Delshad et al,2018) and(Hu, 2018) have been used as medicinal plants to treat various human aliments.

Table 1. Inhibitory effects of 20 plant extracts and ethanol extracts from different parts of Paris polyphylla on Magnaporthe oryzae spore germination.

Twenty extracts with ≥ 10% inhibition rates are listed. Data are Mean± SD.* and **, Statistically significant at< 0.05 and< 0.01,respectively, inDunnett’s multiple comparison test with the negative control (10% ethanol).

In this study, we screened 48 extracts prepared from 8 medicinal plant species using 6 different solvents (Table 1), which resulted in the identification of 20 extracts with ≥ 10% inhibitory activity against(Table 1). These included.(ethanol extract),(petroleum ether,-butanol, ethyl acetate and chloroform extracts),(-butanol, petroleum ether, and ethyl acetate extracts),(-butanol, ethyl acetate and water extracts),(-butanol, petroleum ether and chloroform extracts),(ethyl acetate and water extracts),(-butanol and water extracts), and(petroleum ether and water extracts) (Table 1). The remaining 28extracts had no appreciable inhibitory effect (< 10%, data not shown).

Among the 20 active extracts, 3 extracts exhibited particularly strong inhibitory activity againstspore germination. Similar to the isoprothiolane treatment (Fig. S1-A), with the ethanol (10%) as the negative control (Fig. S1-B), the inhibition rate of the ethanol extract of.plants reached 100% (Fig.S1-C). The ethyl acetate extract of.also showed a clear inhibitory effect (70%), and the-butanol extract of.had an inhibitory rate of 50% (Fig. S1-D).Many round-shaped spores (representative of ruptured spores) were observed in the.and.extract treatments (Fig. S1-C and -D). Thehigh concentrations of the extracts (≥ 25 mg/mL) from.and.were required to achieve an inhibition rate of ≥ 90% (data not shown). The remaining 17 plant extracts showed much lower inhibitory activity against(≤ 30%) (Table 1).

Fig. 1. Antifungal and resistance-inducing activities against rice blast fungus.

A, Increase in blast resistance of rice seedlings by application ofpericarp extract. Four-leaf stage rice seedlings were foliar sprayed withpericarp extract, followed by blast inoculation at 24 h later. Relative fungal growth ofwas examined after 7 d of blast inoculation.

B, Increase in expression levels of rice defense genes (,and) bypericarp extract. Gene expression was examined after 3 d of blast inoculation.

Data are Mean ± SD (= 12). * indicates significant difference from the mock-treated plants (-test,< 0.05).

The-butanol extract ofand ethyl acetate extract ofshowed high antifungal activities (50% and 70%, respectively), but only minor inhibitory effects were observed when these plants were extracted using other solvents (≤ 20%). Further,.has been reported to contain many bioactive substances, such as glycosides, saponins, flavonoids, alkaloids, triterpenoids and steroids (Bonotto et al, 2013; Jia et al, 2017; Wei et al, 2019), which have hypoglycemic, antitumor, antiviral, antifungal and anti-inflammatory effects (Yan et al, 2019). However, only moderate inhibition ofspore germination (30%) was observed with chloroform extract, whereas petroleum ether,-butanol and ethyl acetate extracts showed even lower inhibitory effects (≤ 20%, Table 1). These results are in accordance with previous reports that selection of appropriate extraction solvent(s) is critical for recovery of target bioactive compounds from plants (Dhawan and Gupta, 2017; Randika et al, 2021).

As the ethanal extract of.exhibited strong antifungal activity against(Table 1), we further evaluated extracts from different parts of the.plant. Extracts from various plant parts had significant differences in average inhibition rates. Root and pericarp extracts inhibitedspore germination by 78% and 77%, respectively (Fig.S1-E to -H). In contrast, extracts from the other parts showed moderate inhibition effects (43%–52%) onspore germination. Furthermore, pretreatment of rice seedlings with pericarp extract significantly increased blast disease resistance as shown by reduction ofgrowth compared with the Mock treatment(Fig.1-A).

The roots of.are commonly used to prepare medicinal materials, whereas the pericarps are generally discarded. Only the roots of 3–5-year-old.can be used as medicine, and the slow growth of this plant has led to a shortage of its wild resources (Wang et al, 2019; Ye et al, 2021). Our results indicated that the pericarp can be extracted and used as an antifungal agent, which greatly improved the utilization efficiency of.plants and reduced the cost of Rhizoma Paridis. Many bioactive organic substances have been identified in.extract, including steroidal saponins and their analogs, which have antidepressant, antitumor, antifungal, and immunomodulatory effects (Zhang et al, 2020; Zhao et al, 2020; Thu et al, 2021). It is necessary to identify the molecule(s) in.extract responsible for the inhibition ofspore germination (Table 1) and the induction of rice immune response (Fig. 1) in future studies. Relatedly, it has been reported that saponins inand, and terpenoids inplants are the major corresponding components suppressinggrowth.

We also observed a significant induction of rice defense genes,andin rice seedlings pretreated withextract (Fig. 1-B). These results indicated thatextract activated the host (rice) defense system.encodes a transcription activator and plays crucial roles in benzothiadiazole (BTH)-induced resistance to blast and blight diseases in rice (Shimono et al, 2007).(Agrawal et al, 2000) and(Nakashita et al, 2001) are pathogenesis- related genes that are highly responsive to rice fungi and are widely used as markers of rice defense response.induces cell death via RNase activity in rice, tobacco and(Kim et al, 2011). Hence, these results are consistent with this study that the application ofpericarp extract significantly enhanced blast disease resistance in rice seedlings (Fig. 1-B). Taken together, these results suggested thatextract protected rice seedlings not only by inhibitingspore germination, but also by activating the host (rice) defense system. It has been shown previously that application of plant extracts from,andat 24 h prior to blast inoculation effectively suppressed the development of blast disease. However, whether these extracts are also capable of activation of the host defense system requires further investigation.

In summary, we identified several medicinal plant extracts with considerable potential as new agricultural agents for rice blast control. Especially, theextract not only inhibitedspore germination but also activated the host (rice) defense system. These findings provide a new approach to environmentally safe and sustainable management of plant diseases, as well as the effective utilization of.plants. Nevertheless, some critical questions need to be addressed in future studies, including which chemical component(s) in the.extract are responsible forinhibition and/or host defense activation, and what molecular and physiological mechanisms underpin the beneficial effects of those component(s).

ACKNOWLEDGEMENTS

This study was supported by the National Natural Science Foundation of China (Grant No. 31370306), the Fundamental Research Funds for the Central Universities, South-Central University for Nationalities (Grant No. CZZ22004), and Hubei Province Natural Science Foundation of China (Grant No. 2019CFB804).

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://ricescience.org.

File S1. Methods.

Fig. S1. Inhibition ofspore germination by ethanol extracts from different plant species and parts ofplants.

Table S1. Primer sequences used for qRT-PCR.

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Liu Xinqiong (liuxinqiong@mail.scuec.edu.cn); Jiang Changjie (cypa44@hotmail.com)

17 January 2022;

30 March 2022

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