Powdery mildews (Ascomycota) encompass a category of widespread fungal pathogens that negatively impact a broad range of crops (Glawe, 2008). These fungi are obligate biotrophs, being completely dependent on living tissue for life, and are often host specific, associating with only one or a few species. For example, Blumeria graminis forma specialis tritici (Bgt) is known to be specific to wheat (Triticum aestivum) (Dean et al., 2012; Manser et al., 2021). Although B. graminis f. sp. tritici–resistant wheat cultivars have been developed, complete control of the disease has not been demonstrated and exogenous fungicide application is still necessary (Yang et al., 2008). Unfortunately, decades of application of agrochemical fungicides not only have produced pervasive environmental pollution but also have increased fungicide tolerance among fungal pathogen populations (Gong et al., 2013; Yang et al., 2013). An emerging alternative to the continued overuse of agrochemical fungicides is the adoption of naturally produced molecules, particularly those produced by plant endophytic microbes (Aly et al., 2010; Kharwar et al., 2011).

Endophytic microorganisms, both bacteria and fungi, carry out some part of their life cycle within living plant tissue and cause no disease symptoms (Tan and Zou, 2001; Rodriguez et al., 2009). Although most endophytes are of exogenous environmental original, some may be transmitted vertically in seeds or other generative tissue (Siegel et al., 1995; Rodriguez et al., 2009; Wei et al., 2014). Many endophytes form mutualistic relationships with their hosts, often considerably enhancing growth, defense, and adaptation to stress (Tan and Zou, 2001; Schardl et al., 2004; Rosenblueth and Martínez-Romero, 2006). In addition, endophytes may synthesize their own biologically active products, which can be applied as biocontrol agents either directly or indirectly, to stimulate induced resistance (Staniek et al., 2008; Aly et al., 2010; Kharwar et al., 2011; Vieira et al., 2014). Specifically, in some cases, the presence of endophytic microbes can produce amounts of bioactive secondary metabolites, including hormones, autocoids, and defense-related substances (Tudzynski, 1997; Pirttilä et al., 2004; Tanaka et al., 2005).

Endophytic microbes gained some notability in 1993 with the isolation of taxol, a multifunctional alkaloid, from the endophytic fungus Taxomyces andreanae, although endophytes have been mentioned in the literature since at least 1904 (Stierle et al., 1993; Qian et al., 2014). Since then, several other important compounds have been discovered to be produced by endophytic microbes, including isocoumarin (Findlay et al., 1995), camptothecin (Puri et al., 2005), podophyllotoxin (Eyberger et al., 2006), cochlioquinone A and isocochlioquinone A (Campos et al., 2008), and others (Schulz et al., 2002; Aly et al., 2010; Kharwar et al., 2011; Alvin et al., 2014). In Lasiodiplodia pseudotheobromae F2, isolated from Illigera rhodantha (Hernandiaceae) flower, six sulfureous diketopiperazines, lasiodiplines A–F, were characterized, and lasiodipline E was a potent antibacterial compound against the clinical strains Streptococcus sp., Peptostreptococcus sp., Bacteroides vulgates, and Veillonella parvula, respectively (Wei et al., 2014). In L. pseudotheobromae #1048 AMSTITYEL, isolated from stem of Aegle marmelos, two new compounds, lasdiplactone and lasdiploic acid, were isolated, which showed high xanthine oxidase inhibitory activity. In addition, the endophytic fungus #1048 AMSTITYEL showed maximum in vitro proteolytic and fibrinolytic activity (Meshram and Saxena, 2016; Kumar et al., 2019). In L. pseudotheobromae FKI-4499, isolated from soil collected in Okinawa Prefecture, Japan, aldsulfin, an anti-mannheimiosis agent, was identified, which displayed antibacterial activity against Mannheimia haemolytica and Pasteurella multocida (Sakai et al., 2021). In L. pseudotheobromae IBRL OS-64, isolated from the leaf of Ocimum sanctum, the fungal extract displayed significant antibacterial and anti-biofilm activities against methicillin-resistant Staphylococcus aureus (MRSA) and could be a candidate for antibacterial and antibiofilm drugs (Jalil and Ibrahim, 2021). In L. pseudotheobromae C1136, isolated from Tridax procumbens leaves, the active metabolites produced by the bioherbicidal isolates have bioherbicidal activity against Amaranthus hybridus L. and Echinochloa crus-galli weeds, and rhamnolipid, a biosurfactant produced by the bacterial Pseudomonas aeruginosa C1501, can serve as an adjuvant to improve the penetrability of bioherbicide active components from C1136 for controlling weeds (Adetunji and Oloke, 2013; Adetunji et al., 2017; Adetunji et al., 2018; Adetunji et al., 2020). However, the endophytic L. pseudotheobromae species have not been used in controlling agricultural diseases. Although natural products produced by endophytes are biodegradable, have low environmental toxicity, and show broad-spectrum bioactivity, these microorganisms remain an underutilized resource (Gunatilaka, 2006; Suryanarayanan et al., 2012). Such a class of safe, effective, and environment-friendly biocontrol agents would be particularly well suited for modern integrated management programs, aimed at reducing agrochemical use (Balandrin et al., 1985; Yang et al., 2008).

In our previous work, we isolated the endophytic fungus L. pseudotheobromae LPS-1 from the medicinal plant Ilex cornuta and found that the culture filtrate of the fungal isolate controlled Bgt infection more effectively than the broad-spectrum fungicide triadimefon (10 μg/ml) (Xiang et al., 2016). In this study, we sought to isolate and identify the specific secondary metabolites produced by L. pseudotheobromae LPS-1 that are antagonistic toward B. graminis on wheat. Here, we report the isolation and identification of three such compounds: indole-3-carbaldehyde (A2-5-6-1), indole-3-carboxylic acid (3-ICA; A2-5-6-2), and jasmonic acid (JA; A2-5-6-3). We further demonstrated that JA is antagonistic to appressorium development and haustorium formation during the B. graminis infection process, and 3-ICA synergistically enhances the antagonism of JA against B. graminis on wheat.

In this study, the endophytic fungus L. pseudotheobromae LPS-1 isolated from the medicinal plant, Ilex cornuta, can produce three agriculturally relevant bioactive compounds: indole-3-carbaldehyde, 3-ICA, and JA. Among them, both indole-3-carbaldehyde and 3-ICA are commonly present in microorganisms and plants and present antimicrobial and antitumor activities (Yue et al., 2000; Anderton et al., 2004; Kavitha et al., 2010; Mujahid et al., 2011; Qian et al., 2014; Liu et al., 2017; Zhou et al., 2019; Duong et al., 2021). Some studies have showed that indole-3-carbaldehyde, isolated from Pseudomonas sp. ST4, Streptomyces sp. CT37, Aeromicrobium ponti LGMB491, Janthinobacterium lividum, Clitocybe nuda LA82, Angelica sinensis callus, and Marinomonas sp., has inhibitory effects on the growth of Sporisorium scitamineum, Ustilago maydis, Candida albicans ATCC 10231, Staphylococcus aureus, and Batrachochytrium dendrobatidis JEL215, on zoospore germination of Phytophthora capsici PCM81, on neuroinflammation, and on the biofilm formation of Vibrio cholerae O1, respectively (Brucker et al., 2008; Chen et al., 2012; Rajalaxmi et al., 2016; Gos et al., 2017; Zhou et al., 2017; Liu et al., 2020; Fang et al., 2020). However, other reports about indole-3-carbaldehyde did not indicate its antimicrobial activity against pathogens. In this study, we also demonstrated that indole-3-carbaldehyde has no antifungal activity against Bgt E21 ( Table 2 ). This indicates that indole-3-carbaldehyde may exhibit different extents of antimicrobial activity against different pathogens. Therefore, whether indole-3-carbaldehyde, isolated from L. pseudotheobromae LPS-1, has antimicrobial activity against other plant pathogens will be investigated in the future. For 3-ICA, in plants, it is regarded as an inactive auxin (IAA; indole-3-acetic acid) analog, and IAA is also implicated in plant defense (Bari and Jones, 2009; Borgati and Boaventura, 2011; Petti et al., 2012). Some studies indicate that 3-ICA, a plant cell wall–bound metabolite that could mediate accelerating callose accumulation in response to pathogens, may perform important functions as a mediator of induced resistance in plant basal defense against biotic stress. However, its function may be regulatory or signaling because it has no direct antifungal effect on pathogens (Forcat et al., 2010; Gamir et al., 2012; Iven et al., 2012; Gamir et al., 2014a; Gamir et al., 2014b; Frerigmann et al., 2016; Gamir et al., 2018; Pastor-Fernández et al., 2019). Similarly, in this study, we also found that there was no significant difference in disease severity and pathogenicity of Bgt E21 on susceptible wheat leaves between 3-ICA treatment and the untreated group (Mock) ( Figure 2 ; Table 2 ), which preliminarily indicated that 3-ICA had no inhibitory effect on the infection process of wheat powdery mildew. Contrarily, JA was much more bioactive than indole-3-carbaldehyde and 3-ICA against Bgt E21, and the disease severity of Bgt E21 decreased significantly with the concentration increase of JA treatment ( Table 2 ). In plants, the plant hormone JA and its derivatives are involved in regulating a diverse set of processes including cellular development, defense, and resistance to both abiotic and biotic stress (Ruan et al., 2019; Gomi, 2021). For instance, when applied to plants, the JA-derivative lasiojasmonate A (LasA), isolated from Lasiodiplodia mediterranea, a pathogen of grapes, induces many JA-regulated response in planta at both the genetic and physical level (Chini et al., 2018). The synthetic JA-Ile-macrolactone 5b acts to stimulate induced resistance against pests and pathogens in tea (Lin et al., 2020). In apple trees, JA has been shown to be more effective at stimulating induced resistance to Tetranychus urticae than the fungicide acibenzolar-S-methyl (BTH: benzothiadiazole) (Warabieda et al., 2020). Exogenous treatment with JA can enhance chilling tolerance of peach fruit and reduce the severity of internal flesh browning (Zhao et al., 2021). Some studies have also shown that JA signaling can induce the transcription of several defense-related transcription factors (TFs), such as MYC, MYB, NAC, ERF, and WRKY (Lorenzo et al., 2003; Delessert et al., 2005; Skibbe et al., 2008; Fernández-Calvo et al., 2011; Gao et al., 2011; Qi et al., 2011), and can also induce the MAP kinase cascade (Li et al., 2017), calcium channel activation (Kenton et al., 1999), and other processes that, along with other plant hormones like abscisic acid, salicylic acid, and ethylene, regulate plant growth, development, and stress response (Santner and Estelle, 2009; Ruan et al., 2019). JA is also present in other fungi in addition to L. pseudotheobromae LPS-1, such as Acremonium sp. D212, Botryodiplodia theobromae, L. theobromae strain 2334, and L. iranensis (Husain et al., 1993; Eng et al., 2016; Han et al., 2020; Shen et al., 2022). However, in this study, we do not know the molecular mechanism of 3-ICA and JA antagonizing B. graminis at present. Therefore, whether 3-ICA and JA served as signal molecules to induce the resistance of wheat against the infection of B. graminis will be further elucidated in the future.

Work by others has indicated that 3-ICA can be effectively employed as an antibiotic adjuvant of doxycycline against a range of Gram-negative bacteria (Cadelis et al., 2021). However, 3-ICA as a synergist of JA against plant pathogens has not been reported. Therefore, we wondered whether 3-ICA may interact with JA as an adjuvant to improve its bioactivity against fungal infection. We found that, in this study, there was indeed a synergistic effect between 3-ICA and JA against Bgt E21 on wheat in each of the ratios of 3-ICA to JA (3-ICA:JA) ranging from 1:9 to 9:1. When the compound ratio of 3-ICA to JA was 2:8, the synergistic coefficient was the highest as 22.95 ( Table 3 ). Therefore, we chose the compound ratio of 3-ICA to JA as 2:8 for the following study. Similar to the treatment of JA at 500 μg/ml, there was no symptom on susceptible wheat leaves, and the appressorium and haustorium formation of Bgt E21 was significantly inhibited under the treatment of 3-ICA:JA (2:8) at 40 μg/ml ( Figures 2 , 3 ; Table 4 ). This indicated that treatment with 3-ICA:JA (2:8) at 40 μg/ml was just as affective at controlling Bgt E21 infection as treatment with JA alone at 500 μg/ml, a 12.5× greater concentration of JA. It appears that 3-ICA significantly enhances the antagonistic efficiency of JA against wheat powdery mildew. In addition, to avoid the possibility of pathogenic resistance, antibiotic adjuvants should preferably lack their own antibiotic activity (Bernal et al., 2013), and we found that 3-ICA alone showed no direct activity against Bgt E21. Thus, 3-ICA can be employed as a synergist of JA to enhance the antagonism against B. graminis, and we suggest that the combination of JA and 3-ICA is potentially suitable for modern integrated disease management programs, aimed at reducing agrochemical use. These data further evince the untapped capacity of endophytic microorganisms to contribute useful compounds to agriculture and beyond.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

Conceived and designed the experiments: YQ, LY, and LX. Performed the experiments: YQ, DH, SG, XZ, BY, MX, WS, FZ, ML, TC, DY, LY, and LX. Analyzed the data: YQ, LY and LX. Wrote the paper: YQ, XY, ZW, and LX.

This work was supported by the Natural Science Foundation of China (Grant No. 31701834), the Natural Science Foundation of Hubei Province (Grant No. 2021CFB403), the China Agriculture Research System of MOF and MARA (Grant No. CARS-03), the Youth Science Foundation of Hubei Academy of Agricultural Sciences (Grant No. 2022NKYJJ08) and the Youth Science Foundation of Institute of Plant Protection and Soil Fertility, Hubei Academy of Agricultural Sciences (Grant No. 2021ZTSQJ13). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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