A potential biocontrol agent Streptomyces tauricus XF for managing wheat stripe rust

Abstract

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a devastating disease threatening global wheat production. Biocontrol by beneficial microorganisms is considered an alternative to synthetic fungicide applications. This study aimed to investigate the mechanisms involved in the biocontrol of wheat stripe rust by streptomycetes. A streptomycete strain XF, isolated from the rhizospheric soil of peony, was identified as Streptomyces tauricus based on morphological characteristics and phylogenetic analysis. We determined the inhibitory effect of XF on Pst and biocontrol effect on the disease using XF fermentation filtrate (FL) and actinomycete cell suspension (AC). Results revealed that FL inhibited urediniospore germination by up to 99% and rendered a lethality rate of 61.47% against urediniospores. Additionally, crude extract of ethyl acetate phase of FL caused cytoplasm releases from urediniospores and the deformation of germ tubes. Furthermore, histochemical analyses revealed that treatments of plants with AC and FL increased reactive oxygen species, inhibited haustorium formation, and reduced the biomass of Pst in leaves. Electron microscopy showed that XF mycelium was able to colonize the leaf surface. Moreover, gene expression assays revealed that AC and FL treatments induced the expression of a number of pathogenesis-related genes in wheat leaves. Besides, in the greenhouse experiments, the control effects of AC and FL reached 65.48% and 68.25%, respectively. In the field, application of XF fermentation broth significantly reduced the disease indices of stripe rust by 53.83%. These findings suggest that XF is a potential biocontrol agent for managing wheat stripe rust disease.

Background

Wheat stripe rust caused by the obligate parasitic fungus Puccinia striiformis f. sp. tritici (Pst) is a devastating foliar disease that has historically caused significant yield losses worldwide. It has become the greatest biotic constraint to wheat production in the twenty-first century, resulting in annual losses of one billion dollars worldwide (Beddow et al. 2015; Schwessinger 2017). China is the largest wheat producer and is the largest stripe rust endemic region in the world (Ye et al. 2019). China’s epidemiological pattern of stripe rust is more complex and variable than in other countries, making disease control more difficult (Ye et al. 2019). To effectively manage wheat stripe rust, an integrated control strategy must be adopted based on planting disease-resistant cultivars, supplemented by fungicides and cultivation management measures. However, most significant genetically resistant varieties gradually lose their resistance after 3 to 6 years of field cultivation due to the rapid Pst virulence mutations (Han et al. 2015). Additionally, the continual and indiscriminate use of fungicides causes environmental pollution and the development of more aggressive isolates as they acquire resistance against routinely used fungicides (Lucas et al. 2015). Therefore, there is an urgent need to develop new strategies for better disease control.

In the recent era of biologically sustainable agriculture, biocontrol agents (BCAs) for preventing crop diseases are receiving considerable attention, and BCAs have emerged as a promising measure for management of wheat stripe rust. For example, Cladosporium cladosporioids and Alternaria alternate inhibit urediniospore germination via parasitism (Zhan et al. 2014; Zheng et al. 2017). Bacillus subtilis E1R-j has been shown to effectively control wheat stripe rust by causing urediniospores and germ tubes to rupture, releasing protoplasm, and causing fungal structures to malfunction (Li et al. 2013). Endophytic Paneibacillus xylanexedens 7A and B. megaterium 6A induce wheat resistance and reduce the disease severity of stripe rust with efficacy of 61.11% and 65.16%, respectively (Kiani et al. 2021). Recent studies reveal that Streptomyces spp. have excellent biological control efficacy against various wheat fungal pathogens. S. pratensis S10 was reported to significantly inhibit the growth of Fusarium graminearum and effectively control wheat scab and decrease deoxynivalenol content (Zhang et al. 2021). Two BCAs, S. neyagawaensis and S. viridosporus, decrease the severity of wheat leaf rust and the number of pustules (Sub et al. 2004; Araujo et al. 2019). In a greenhouse pot trial, plants treated with seed coatings of either S. hygroscopicus or S. lydicus had > 24% lower disease severity than control plants infected with F. pseudograminearum (O'Sullivan et al. 2021). Several Streptomyces spp. members have been recognized as diazotrophic endophytes that protect wheat against Rhizoctonia solani attack by direct antagonism and induction of plant defenses (Patel et al. 2018).

Streptomycetes can suppress or kill microbial opponents by producing bioactive molecules, such as enzymes disrupting fungal cell walls and compounds with fungicidal activity (Gupta et al. 1995; Diaz-Cruz et al. 2022). Biocontrol activity often occurs before the pathogens completely infect their respective hosts. It has been shown that foliar sprays of bioactive compounds produced by Streptomyces spp. can prevent symptoms caused by microbial pathogens, although Streptomyces spp. inhabiting plant leaves are rare (Han et al. 2021). Additionally, Streptomyces spp. have been shown to trigger localized and systemic resistance in plants and to inhibit pathogen growth via induction of plant defense mechanisms (Lehr et al. 2008). In general, induced resistance can be divided into systemic acquired resistance (SAR) and induced systemic resistance (ISR). The induction of plant defenses by Streptomyces species differs from traditional ISR, as it involves features of both ISR and SAR, as well as cross-talk between many different phytohormone signaling pathways (Newitt et al. 2019). In any case, the ability of Streptomyces spp. to act as inducers of systemic plant resistance renders them promising candidates for effective biocontrol of plant diseases.

However, so far, there are few reports regarding the utilization of Streptomyces spp. for controlling wheat stripe rust. In this study, S. tauricus strain XF was screened as a potential BCA and displayed high antagonistic activity against Pst. This study aims to define further the biocontrol abilities of XF against wheat rust to advance the development of new biological fungicides.

Results

Streptomyces isolate XF suppressed wheat stripe rust disease

The Streptomyces isolate XF with strong inhibitory activity against Pst urediniospores was isolated from the peony rhizosphere soil collected in Yangling, Shaanxi, China. Light microscopy revealed abnormal germination of urediniospores treated with various dilutions of XF fermentation filtrate (FL) (Additional file 1: Figure S1). Among them, urediniospores treated with the original FL (1×) failed to germinate (Additional file 1: Figure S1), which translates into an inhibition rate of 99.17% (Fig. 1a). The 100-fold dilution of FL (100×) showed antagonistic activity against urediniospore with a 69.68% germination rate, which was considerably lower than the blank control (CK) (Fig. 1a). Meanwhile, the original FL showed a lethality rate of 61.47% against urediniospores, while triadimefon (Tr) was 95.78% (Additional file 1: Figure S2b, c). Electron microscopy results showed that most urediniospores remained non-germinated on wheat leaves sprayed with FL and Tr (Additional file 1: Figure S2d). These results suggest that FL has strong inhibitory effects against Pst urediniospores.

Fig. 1

The control effect of Streptomyces tauricus XF on wheat stripe rust. a The germination rate of urediniospores treated with various dilutions of fermentation liquid. SDW, sterile distilled water; Tr, triadimefon was 20 μg/mL; FL, XF fermentation filtrate; ‘ × ’ represents the dilution factor. b The disease index in a pot trial. Tr, 20% triadimefon emulsions at a concentration of 0.5 g/L. c Control efficiency of XF against wheat stripe rust in a pot trial. The three values were not significantly different from each other. d The disease index of wheat stripe rust in the field trial. e Wheat leaves disease incidence in the field trial. f The control efficiency in the field trial. SDW, sterile distilled water; AC, XF actinomycete cell suspension (10⁷ CFU/mL); FL, XF fermentation filtrate; XF, XF fermentation broth containing AC and FL. Values in charts are mean ± SD. By Duncan’s new multiple range tests, different lowercase letters indicate differences with significance level 0.05

The actinomycete cell suspension (AC, 10⁷ CFU/mL, Additional file 1: Figure S3) and the original FL were used to spray 14-day-old seedlings in the pot inoculation experiments. As shown in Additional file 1: Figure S4a, b, in the control treatment, Pst inoculated leaves were chlorotic and covered with urediniospores, whereas urediniospores were only observed sporadically on leaves of plants in the treated groups. XF AC and FL treatments significantly decreased the stripe rust disease indices (Fig. 1b) and resulted in disease control efficiencies being 65.48% and 68.25%, respectively (Fig. 1c).

In field trials, wheat leaves spray-inoculated with sterile distilled water (SDW), Tr, and XF fermentation broth (XF) on April 21 and April 26, respectively, and the incidence of wheat stripe rust was investigated two weeks later. The results showed that plants in control groups were moderately susceptible, with abundant urediniospores and no tissue necrosis. Wheat treated with XF appeared more resistant, with lower levels of sporulation and chlorotic stripes on the leaves (Additional file 1: Figure S4c, d). Overall, the disease indices and incidence of diseased leaves in the plots treated with XF were significantly lower than in the control treatment (Fig. 1d, e). Strain XF elicited a biocontrol efficacy of 53.83% against stripe rust (Fig. 1f).

Characterization and identification of the strain XF

Strain XF exhibited typical morphological characteristics of the genus Streptomyces. The XF colony had a hard texture and a wrinkled surface with white aerial mycelia and light pink spores on Gause’s Medium (GM) (Fig. 2a). Different spore masses were present on various agar plates (Additional file 1: Figure S5c). The details of the XF cultural characteristics are shown in Additional file 2: Table S1. Moreover, scanning electron microscopy (SEM) photos revealed the morphology of long filamentous rods of the XF hyphae and the presence of cylindrical spores, forming a flexuous spore chain with a rough surface (Fig. 2b). Biochemical tests indicated that XF was able to use all the tested carbon and nitrogen sources (Additional file 1: Figure S5a, b and Additional file 2: Table S2). Extracellular enzymatic activity assays revealed that strain XF could secret several hydrolytic enzymes, including amylase, cellulase, and protease (Additional file 1: Figure S5d). Additionally, XF was able to grow at low temperatures (Additional file 1: Figure S5e). Although its growth rate slowed with decreasing temperature, it maintained viability at 9°C and 16°C, the culture conditions for Pst.

Fig. 2

Identification of XF. a The morphological feature of XF on Gause’s Medium (GM). Bar = 1 cm. b Scanning electron microscope photo of XF spore chain. Bar = 5 μm. c Phylogenetic tree based on the partial nucleotide sequence of 16S RNA genes. Numbers between segments represent bootstrap support values, and ‘T’ above represents the strain as a model strain

To observe the colonization of XF on leaves at low temperature, AC (1 × 10⁷ CFU/mL) were spray-inoculated onto wheat leaves. Electron microscopy shows the XF growth in wheat leaves treated with AC for 5 days at 16°C (Additional file 1: Figure S6). We observed a mass of superficial mycelia covering the leaf surfaces (Additional file 1: Figure S6b), and some appeared to grow into the stomatal openings (Additional file 1: Figure S6c) and substomatal cavities (Additional file 1: Figure S6d). These results indicate that XF could grow on wheat leaves.

To confirm the phylogenetic relationship between XF with Streptomyces species, we amplified the 16S rRNA sequences of XF and conducted sequence alignments in the EzTaxon-e server. The original 16S rDNA sequencing data were available in the NCBI SRA database with the accession number OM541329. Analysis of the 16S rRNA sequence demonstrated that XF matches S. tauricus JCM 4837 (AB045879) at 99.65% and that XF and S. tauricus clustered in one phylogenic group (Fig. 2c). These results confirm that strain XF is an isolate of S. tauricus.

Strain XF inhibited Pst growth in leaves

The primary leaves of 14-day-old wheat were inoculated with 20 µL fresh urediniospores of Pst (3 × 10⁴ spores/mL). The AC (10⁷ CFU/mL) and FL of XF was sprayed onto the wheat leaves 24 h before Pst inoculation, respectively. Leaves were then harvested at 36 and 72 hours post-inoculation (hpi) of XF, and the fungal structures were stained with wheat germ agglutinin (WGA). The results showed that the infected area, hyphal length, and haustoria of Pst at 36 hpi and 72 hpi on AC- and FL-treated leaves were considerably lower than those in control (SDW) (Fig. 3a–d). In particular, haustorium formation rates was reduced more significantly at 72 hpi comparison with at 36 hpi (Fig. 3d). Meanwhile, the effects of AC and FL treatments were comparable to those of the Tr treatment. These observations demonstrated that AC and FL strongly reduced the Pst infection areas, hyphal lengths, and haustorium formation rates. In addition, the Pst biomass in wheat was also significantly reduced at 72 h after treatment with AC and FL (Fig. 3e). Thus, we conclude that strain XF limits Pst expansion in wheat leaves.

Fig. 3

Effects of XF on Pst growth in leaves. a Histological observation of fungal growth in wheat. SV, substomatal vesicle; IH, infectious hyphae; HMC, haustorial mother cell. Bars = 20 μm. b Infection area, the average area of the expanding hypha plus the host cells, was calculated by CellSens Entry software. c Hyphal length is the average distance from the junction of the substomatal vesicle and the hypha to the tip of the hypha. d Haustoria formation rate of Pst with various treatments. e RT-qPCR analysis of the relative expression levels of PstEF in leaves at 72 h after inoculation with Tr, AC, and FL. TaEF and PstEF were used to normalize the RNA level of wheat leaves and Pst, respectively. SDW, sterile distilled water; Tr, 20% triadimefon emulsions at a concentration of 0.5 g/L; AC, XF actinomycete cell suspension (10⁷ CFU/mL); FL, XF fermentation liquid without actinomycete cells. Values in charts are mean ± SD. An asterisk indicates significant differences based on the unpaired two-tailed Student’s t-test with the P values marked (*P < 0.05; **P < 0.01) between the SDW treatment and other treatments

Strain XF promoted ROS burst in wheat leaves and reduced Pst infection rates

To further define the repressive role of XF on Pst, fourteen-day-old wheat were inoculated with AC and FL, and maintained under normal growth conditions for 24 h, then inoculated with Pst. Leaves were collected at 36 hpi and 72 hpi of XF, and DAB staining were performed to detect the reactive oxygen species (ROS) area and stripe rust infection. After Pst infection, ROS accumulation were observed in Tr, AC, and FL treatment (Fig. 4a). The ROS areas in the AC and FL treatments were considerably more significant than the water control at 36 h and 72 h (Fig. 4a, b), indicating that AC and FL promoted ROS bursts in wheat leaves (Fig. 4). The infection rates of Pst were lowered more by XF compared to SDW, as shown in Fig. 4c. AC treatments resulted in significantly lower infection rates than SDW treatments, 1.83% at 36 h and 2.29% at 72 h. At 36 h, FL had a strong inhibitory effect on Pst infection with only a 3.90% infection rate.

Fig. 4

Effects of XF treatments on ROS production and infection rate of Pst on wheat leaves. a Histological observation of H₂O₂ accumulation after DAB staining in wheat leaves. SV, substomatal vesicle. Bars = 20 μm. b, c The ROS area of wheat leaves (b) and the infection rate of Pst (c) in the various treatment groups. Only infection sites where substomatal vesicles had formed over stomata were considered successful infections and were microscopically observed for the presence or absence of haustoria. The infection rate was calculated as the number of penetration sites that exhibited one or multiple haustoria in relation to the total number of stomata. Values in the chart are mean ± SD. By Duncan’s new multiple range tests, different lowercase letters indicate differences with significance level 0.05

Strain XF induced the expression of defense-related genes in wheat leaves

To determine whether the XF strain could induce disease resistance in wheat, the first true leaf of ‘2 leaves and 1 heart’ wheat was sprayed with FL and AC (10⁷ CFU/mL) and the second true leaves were collected at 12, 18, 24, 36, 42, 48, 72, and 120 hpi of XF. The transcript levels of genes encoding phenylalanine ammonia-lyase (PAL) and pathogenesis-related (PR) proteins, including antifungal protein (PR1), β-1,3-endoglucanases (PR2), chitinases (PR3), endochitinase (PR4), and peroxidase (PR9) were analyzed. As shown in Fig. 5, six genes were upregulated to varying degrees at different time points except for the 18 hpi. Salicylic acid (SA)-related genes PR1, PR2, and PAL showed maximum expression at 120 hpi in FL-treated plants, with a fold change of 134.18, 38.04, and 3.24, respectively, compared to the control (Fig. 5 and Additional file 1: Figure S7). However, the peak of relative expression of PR1 and PR2 in AC-treated plants appeared at 12 hpi, with 7.30- and 7.97-fold change, respectively. These results indicated that the expression of gene PR1 and PR2 were mainly activated by AC at the early stage of inoculation, while FL mainly induced the expression of the two genes in the late stage. Additionally, with the jasmonic acid (JA)/ ethylene (ET)-related genes PR3, PR4, and PR9, the overall dynamics of PR3 expression were similar to those with PR4 expression (Fig. 5). Expression levels of the two genes in the FL-treated group were all higher than those in the AC-treated group at 12, 42, 48, and 120 hpi. Remarkably, the relative expressions of gene PR3 and PR4 both reached a maximum at 120 hpi, and the expression of PR4 in the FL-treated group was tenfold higher than in the AC-treated group at this time (Additional file 1: Figure S8a, b). For the gene PR9, wheat treated with AC or FL, the gene was significantly up-regulated at all time points compared to the control group, except for 18 hpi and 36 hpi (Fig. 5). Gene expression peaked at 48 h in both AC- and FL-treated groups, with 44.36- and 375.55-fold increases (Additional file 1: Figure S8c), respectively.

Fig. 5

Expression of defense-related genes in wheat leaves induced by XF. The first true leaf of wheat was sprayed with fermentation filtrate (FL) and actinomycete cell suspension (AC), and the second true leaves were collected at 12, 18, 24, 36, 42, 48, 72, and 120 hours post-inoculation (hpi) of XF. The horizontal axis in heat maps represents different time points, and each square shows the relative transcript levels of each gene in the AC or FL treatment. The heatmap was row-normalized using the scale method. The higher the gene expression, the hotter (redder) the color. An asterisk indicates significant differences based on the unpaired two-tailed Student’s t-test with the P values marked (*P < 0.05; **P < 0.01) between treatments and the sterile distilled water (SDW) control, and only significantly up-regulated expressions are marked in the figure

The ethyl acetate extracts of FL exhibited antagonistic activity against urediniospores

To obtain the active ingredients, XF FL was extracted sequentially using petroleum ether (PE), dichloromethane (DCM), ethyl acetate (EA), n-butanol (NB), and the remaining portion (R) was concentrated. The EA phase of FL had the highest Pst antifungal activity and the urediniospores germination rate was reduced to 0.66% (Additional file 1: Figure S9). Thus, subsequent tests were conducted with EA as an extraction solvent, and the ethyl acetate phase active substance (EAP) was obtained by extraction with EA. After extraction of the FL with EA, the remaining aqueous fraction were concentrated in vacuo to obtain the non-ethyl acetate phase active substance (NEAP). Consistent with the results above, as shown in Additional file 1: Figure S10, the EAP active substance exhibited a much stronger antagonistic effect than the NEAP. The germination rates of urediniospores treated with NEAP were only 16.82%, whereas the EAP-treated urediniospores failed to germinate (Additional file 1: Figure S10), which translates into an inhibition rate of 99.90%, and proteinase K (proE K) treatment did not affect the inhibitory activity of the extracts (Fig. 6a and Additional file 1: Figure S10). These results indicate that the active substance reducing urediniospore germination in XF FL is probably not a protein(s) but a chemical or combination of chemicals with polarity similar to the EA.

Fig. 6

Effect of different concentrations of EAP solution on the germination of Pst urediniospores. a Comparison of the inhibition rate of urediniospore germination by EAP and NEAP before and after treatment with proE K. Values in the chart are mean ± SD. By Duncan’s new multiple range tests, different lowercase letters indicate differences with significance level 0.05. b Inhibition rate of urediniospore germination by incubating with different concentrations of EAP solution for 12 h at 9°C in the dark. c Morphology of urediniospores in the above treatments under the optical microscope. Bars = 50 μm. EAP, ethyl acetate phase active substance; NEAP, non-ethyl acetate phase active substance; proE K, proteinase K. Sterile distilled water (SDW), 1% methanol, and Tr were used as controls

To confirm the inhibitory effect of EAP extracts on Pst, the antifungal activities of EAP at various concentrations were evaluated by testing the urediniospore germination and Pst mortality rates. The inhibition rate of urediniospore germination was 18.29% at 25 mg/L, but increased significantly to 86.12% at 50 mg/L (Fig. 6b). The inhibitory effect of EAP on urediniospores was up to nearly 99%, similar to that of the Tr treatment, when the concentration was increased to over 100 mg/L. As shown in Fig. 6c, low concentrations of EAP resulted in urediniospore cytoplasm release and germ tube swelling and deformation, accompanied by much shortened germ tube than observed in the SDW treatment. The results of the survival tests showed that EAP treatment resulted in the death of urediniospores (Fig. 7a). The mortality rate increased with increasing EAP concentrations and duration of treatments, and eventually reached 25%, which was much higher than the SDW treatments but still far less than the Tr-treated group (Fig. 7b).

Fig. 7

Mortality of ethyl acetate phase active substance (EAP) on Pst urediniospores. a Status of urediniospores after incubation at 9°C in the dark for different times in each treatment under the optical microscope. AS, alive status; ES, emergence status; DS, death status. Bars = 50 μm. b Urediniospore mortality rate in the various treatment groups at each time point. Comparisons were made between different time points for the same treatment, and the values in the chart are mean ± SD. By Duncan’s new multiple range tests, different lowercase letters indicate differences with significance level 0.05

Discussion

Using microorganisms to control plant disease is a sustainable alternative to conventional fungicides (Araujo 2022). The ability of Streptomyces species to produce plant-protective substances, such as enzymes, secondary metabolites, and volatile organic compounds, as well as their ability to induce plant immunity to respond to pathogens rapidly, indicate that they are good candidates as biocontrol agents (Newitt et al. 2019). In this study, S. tauricus XF was demonstrated to effectively control wheat stripe rust and its secondary metabolites have positive roles in resistance to Pst. The mechanisms behind this phenomenon may be associated with multiple factors, such as direct phytopathogen suppression and inducing broad-spectrum plant disease resistance. Both mechanisms have potential applications for biological control of wheat stripe rust.

Five types of spores are produced during the stripe rust fungus life cycle; asexual urediniospores are responsible for facilitating the most damaging phase of wheat stripe rust disease (Jiao et al. 2017), and almost all biochemical and molecular studies have focused on urediniospores and their infection structures (Chen et al. 2014). Consequently, urediniospores will likely play a decisive role in developing more effective strategies for stripe rust control. Screening beneficial microorganisms for agents that can suppress urediniospores has been suggested in previously reported literature (Li et al. 2013; Zhan et al. 2014; Zheng et al. 2017) and has been confirmed in our research. The inhibition of urediniospore germination was used as an indicator to evaluate the antagonistic activity of candidate isolates against Pst. The strain XF stood out because of its strong inhibitory effect on urediniospores. The mortality rate of urediniospores was used as a marker for the fungicidal activity of the XF strain against stripe rust. Trypan blue staining was used to observe activities of urediniospores during interaction between XF and Pst (see dark blue deactivated urediniospores in Fig. 7). Based on these observations, we believe it is possible to combine studies on the germination inhibition rate and mortality of urediniospores to screen microorganisms for bio-control potential against wheat rust more efficiently.

S. tauricus is a microorganism of great interest in the medical field by secreting various compounds with anti-cancer and anti-human pathogenic activity, but it has been less studied in agriculture. However, as early as twenty years ago, S. tauricus 19/97 M was found to stimulate the growth of coniferous seedlings and protect them from diseases caused by Fusarium and Alternaria fungi (Gaidasheva et al. 2022), which heralded the potential of S. tauricus for agricultural disease control. This potential was subsequently demonstrated in several reports. Berg et al. (2006) found that a strain identified as S. tauricus isolated from strawberry rhizosphere soil could inhibit the growth of Verticillium dahlia, which causes yellow wilt. The S. tauricus JS2 strain has antagonistic activities against Ceratocystis fimbriata, the pathogen of pomegranate wilt, and inhibits southern root-knot nematode infestations (Zhou et al. 2016). However, studies on the effectiveness of S. tauricus against phytopathogenic fungi have been limited to determining inhibition activities in vitro or in greenhouse experiments (Bhai et al. 2018). There is little information in the literature about the efficiency of this genus against plant fungal diseases under field settings. In the present study, S. tauricus XF has high efficacy against stripe rust in greenhouse and field trials. Interestingly, XF mycelia could grow on wheat leaf surfaces and XF perform cell wall hydrolytic activities and the ability to utilize various carbon sources in plate assays. These results indicate that S. tauricus XF has potential for agricultural applications to control foliar disease in wheat.

Streptomyces spp. are known to be producers of antifungal compounds (Sabaratnam and Traquair 2015). Numerous studies indicate that the EAP of metabolites secreted by Streptomyces spp. have a broad spectrum of antifungal activities. For example, the EAP of S. griseocarneus was able to inhibit the growth of F. oxysporum f. sp. cubense (Jakubiec-Krzesniak et al. 2018), Colletotrichum gloeosporioides, and the rice pathogen Burkholderia gladioli (Betancur et al. 2017). The antifungal metabolites of S. alfalfa XN-04 were also concentrated in EAP extracts (Chen et al. 2021). In this study, the XF EAP had potent inhibitory activity against Pst. EAP begins to work within the first 2 h of Pst interactions. Although low concentrations of EAP did not affect urediniospore germination, they did impact germ tube growth by suppressing length extensions or abnormal branching (Fig. 6c), as shown in leaf observations (Fig. 3a). Higher concentrations of EAP exerted biocontrol activity by inhibiting germination and killing the urediniospores. Our findings support literature reports on the antifungal activity of Streptomyces spp. For instance, S. griseocarneus could inhibit the spore germination and mycelia growth of tomato fungal pathogens by secretion of rhizostreptin (Sabaratnam and Traquair 2015). Streptomyces sp. CEN26 produces 2,5-bis (hydroxymethyl) furan monoacetate, a compound that deformed conidial germination and appressorium formation of the fungal pathogen (Phuakjaiphaeo et al. 2016). It is possible that S. tauricus XF tested herein synthesizes extracellular antifungal compounds antagonistic to Pst.

An additional mechanism whereby Streptomyces spp. can indirectly protect plants is by activating host resistance pathways. Streptomyces spp. have previously been shown to activate plant defense responses generated by biotic stresses through the induction of antioxidant enzymes and improved ROS management (Singh et al. 2016). Previous studies speculated that ROS was involved in initiating resistance responses in potato tubers or by acting as an intermediate transducer of elicitor signaling (Zhao et al. 2005). In our study, the ROS areas of leaves at 24 h after XF application were significantly more extensive than SDW at 12 h and 48 h after Pst inoculation (Fig. 4), indicating that XF enhances ROS generation in wheat. We speculate that increased defense responses occurred after XF inoculation. Streptomyces spp. have also been shown to induce the expression of defense associated genes in plants (Vurukonda et al. 2018). Gene PR1 has commonly been used as a SAR activation marker, which enhances plant resistance to various pathogens (Ali et al. 2018; Jia et al. 2022). In this study, FL significantly induced PR1 gene expression. A similar phenomenon was also reported in several other studies. For example, the fermentation broths of Streptomyces sp. JCK-6131 (Le et al. 2021) and Streptomyces sp. AgN23 (Vergnes et al. 2020) were previously reported to induce PR1 gene expression in tomato and Arabidopsis thaliana. Also, strain FL and AC were able to activate the expression of the PR3, PR4, and PR9 genes. Many studies have shown that Streptomyces spp. can trigger ISR by activating defense pathways dependent on either SA or JA/ET. Van Wees et al. (2008) reported that activation of the SA and JA/ET pathways resulted in enhanced protection compared with independent activation of each pathway. Indeed, priming oak roots with Streptomyces AcH 505 protected the trees against powdery mildew by activating both the SA and JA/ET signaling pathways (Kurth et al. 2014). In comparison, streptomycetes appeared to activate plant defense response pathways without pathogen infections (Kurth et al. 2014). However, whether the XF-induced plant disease resistance is dependent on both SA and JA signaling pathways needs to be further studied.

Conclusions

The effectiveness of the isolated S. tauricus strain XF in controlling the wheat stripe rust disease was investigated in this research. Strain XF FL exhibited strong antagonistic and fungicidal effects against urediniospores. In particular, the EAP of FL has been shown to possess high antifungal activity, which provides a theoretical foundation for the extraction and isolation of antimicrobial compounds in future studies. Additionally, both FL and AC have been shown to hamper Pst growth in leaves and enhance ROS production in wheat, which plays important roles in reducing the incidence of diseased leaves and disease index. Furthermore, XF fermentation broth appeared to reduce stripe rust development in the field. However, additional research is needed to determine how XF colonizes leaves and whether the XF colonization induces wheat disease resistance against stripe rust.

Methods

Plant material, plant pathogens, and chemical fungicide

The wheat genotype Suwon 11 and the Pst pathotype Chinese Yellow Rust 31 (CYR31) were used in laboratory and greenhouse experiments. Both were obtained from the Crop Disease Prediction and Management Group at Northwest A & F University (Yangling, Shaanxi Province, China). Wheat seedlings were grown in a greenhouse under 8/16 h night/day conditions at 16°C. Pst race CYR31 was propagated on Suwon 11 as described by Tang et al. (2015b). Fresh urediniospores were harvested from infected wheat leaves and stored at 4°C with desiccant until used. Tr, 20% Emulsifiable Concentrate (Jiangsu Jianpai Agrochemical Co., Ltd.) at a concentration of 0.5 g/L was used as a positive control in pot trial and field trial.

Isolation of bio-control strains and preparation of formulations

The healthy peony rhizosphere soil was used to isolate and screen the potential strains for biocontrol. Healthy plant roots were collected from soil in Yangling, Shaanxi, China (108°4′9″E, 34°15′49″N), and the surface soil was gently brushed off. Rhizosphere actinomycetes were isolated using dilution plating (Awla et al. 2017). Actinomycetes colonies were chosen based on their growth rate, colony size, and morphological traits on the agar plates. Pure cultures were obtained by re-growth on GM plates and preserved as spore suspensions in 20% glycerol in a  −80°C freezer.

To obtain fermentation broth, 5 plugs (Ф = 6 mm) of the isolates were placed into a 250 mL conical flask containing 100 mL liquid GM and shaken at 28°C and 160 r/min for 7 d. After centrifuging at 10,000 g for 10 min, the actinomycete cells at the bottom, containing XF mycelial fragments and spores, were re-suspended with SDW and adjusted to a concentration to 10⁷ CFU/mL of AC. The supernatant was filtered through a 0.22 μm sterile filter to obtain FL without actinomycete cells.

Evaluation of biocontrol efficiency

In vitro antifungal activity test

For screening strains with antagonistic effects on Pst, the inhibitory effect of the isolate’s FL with different dilutions on Pst urediniospores germination was determined according to previously described (Li et al. 2013), with slight modifications. Briefly, the isolate’s FL were collected and diluted with SDW to obtain a stock solution and 10-, 100-, and 1000-fold dilutions. The positive control was 20 μg/mL Tr and the negative control was SDW. Each treatment consisting of 20 mL of the above solutions was added to a 9-cm-diameter petri dish and freshly harvested urediniospores were uniformly sprinkled on the liquid surface. All treatments were incubated for 12 h at 9°C in the dark and urediniospore germination was evaluated with a light microscope (Olmypus BX-43). A spore was considered to have germinated if the length of the germination tube was at least half the length of the spore.

For the lethality assay, the urediniospore survival status in FL solutions was investigated, with SDW as a blank control and 100 μg/mL Tr as a positive control. The assay was performed as described above and samples were kept in the dark at 9°C. After 12 h, urediniospores were collected and stained with trypan blue as previously described, with slight modifications (Swain et al. 2017). Briefly, the harvested urediniospores were stained with 0.2% trypan blue for 3 min at 25°C. The staining was observed, and spore survival rates were calculated. The experimental designs were completely randomized with three replicates for each treatment and repeated three times.

Pot assays

The biocontrol efficacy of XF against stripe rust in growth chambers was determined with the wheat cultivar Suwon 11. For the pot-based inoculation assays, 14-day-old seedlings were sprayed with FL and AC (10⁷ CFU/mL), respectively, using a small air hand sprayer. The fungicide Tr (0.5 g/L) and SDW were positive and negative controls, respectively. At 24 h after treatment, the leaves were inoculated with 20 µL of Pst urediniospores at a concentration of 3 × 10⁴ spores/mL suspended in the e-fluoride solution. The inoculated seedlings were held below dew point humidity conditions at 10°C in the dark for 24 h, and then moved to the greenhouse regimen at 16°C. Three pots each with 9 wheat seedlings were used for each treatment, and the experiments were repeated three times. After 14 d of incubation in the growth chamber, the severity (S) of the wheat stripe rust infections was recorded. Based on the percentages of the leaf areas covered by urediniospores, the number of diseased leaves were recorded at all levels and the incidence (I), average severity ($\overline{S }$), disease index (DI), and control effects (CE) were calculated as follows:

$$\mathrm{I}({\%})=\frac{\text{number of diseased leaves}}{\text{number of investigated leaves}}\times 100{\%}$$

$$\overline{S }(\%)=\frac{{\sum }_{i=1}^{n}({X}_{i}\cdot {S}_{i})}{{\sum }_{i=1}^{n}{X}_{i}}\times 100$$

$$DI=\frac{{\sum }_{i=0}^{n}({X}_{i} \cdot {S}_{i})}{{\sum }_{i=0}^{n}(X \cdot {S}_{max})}\times 100$$

$${CE}_{treatment}(\%)=\frac{{DI}_{control}-{DI}_{treatment}}{{DI}_{control}}\times 100\%$$

Severity was classified at 1 to 8 levels corresponding to a coverage of 1, 5, 10, 20, 40, 60, 80, and 100% according to the percentage of leaf areas covered by the urediniospores. i indicates the disease grade, X_i consists of the number of leaves with disease grade i, S_i is the value of disease grade i; X represents the total number of investigated leaves, while S_max shows the highest severity grade; treatment indicates different treatments, control indicates blank control, CE_treatment is the control efficiency of different treatments (These classifications are defined by NY/T 1443.2–2007, the agricultural industry standard of the People's Republic of China).

Field trials

Wheat cultivar Mingxian 169 was planted for field experiments at Caoxin Village, Yangling, Shaanxi Province in China, during the 2021–2022 growth season. The field trials were conducted using a randomized plot design with three replicates for each treatment. Each plot was 5 m² in size with 20 cm spaces between the rows and 20 cm between the plots. Seeds were sown on October 23, 2021, and the field was managed using conventional practices in the region. At elongation stage of wheat with no rust uredia observed, treatments of XF fermentation broth and Tr were applied on 21st and 26th of April 2022. Strain XF fermentation broth containing actinomycete cells and fermentation filtrate was used, and the Tr was applied at a 0.5 g/L concentration. SDW was a negative control. Each plot was sprayed with 600 mL each of the above solutions. On May 11, 2022, 250 leaves were surveyed in each plot, and the incidence and severity of wheat stripe rust under each treatment was recorded.

Identification and characterization of XF

Morphological and cultural characterizations

Morphological characterizations were conducted by SEM (Hitachi model S-3400N, Japan). The cultural characteristics of XF, including color of spore mass, aerial mycelium, substrate mycelium, and diffusible pigments, were recorded after incubation at 28°C for 7 days on TSA, YMS, 2CMY, AS-1, ISP3, ISP4, and MS media (Kieser et al. 2000), and ingredients of culture media are in the Additional file 2: Table S3.

Phylogenetic analyses

The forward primer (5’-ATGCCATTCGTGCGGAGGTTG-3′) and the reverse primer (5’-CGTCTCTGCTGTCATCACTTCGTAT-3′) were used for 16S rRNA sequence amplification (Zhang et al. 2021). The amplifying procedure followed the method described by Jangir et al. (2018), and the amplified 16S rRNA fragments were sequenced (Qingke, Xi'an, China), and submitted to the EzTaxon-e server (http://www.ezbiocloud.net/) for comparative analysis. Phylogenetic trees were constructed with MEGA 7.0 software using the Maximum Likelihood method with 1000 bootstrap replications (Kumar et al. 2015).

Strain characterizations

According to the Manual of Common Bacterial System Identification, six different carbon substrates (sucrose, rhamnose, mannitol, lactose, glucose, and xylose) and the nitrogen sources (Ca (NO₃)₂, KNO₃, NaNO₃, NH₄Cl, NH₄NO₃, and glutamate) were used to substitute for the original carbon and nitrogen sources resulting in a final carbon source concentration of 1% and a nitrogen source concentration of 0.5%. The XF strain was inoculated on medium plates with different carbon and nitrogen sources, with the nitrogen source basal medium and the carbon source basal medium serving as controls. The characteristics of the colony mycelium, and the colony color were observed after 7 days of incubation at 28°C. To test XF growth at low temperatures, AC were inoculated onto GM plates and incubated separately at 9°C, 16°C, and 28°C. Mycelial growth was recorded on the 5th day after inoculation. In addition, XF was examined qualitatively for the production of extracellular enzymes, including protease, cellulase, and amylase, according to previous protocols (Hu et al. 2022).

ROS staining and measurements of infection rate

H₂O₂ accumulation was detected by staining leaves with 3, 3′-diaminobenzidine (DAB) (Xu et al. 2019). Briefly, leaves collected 36 h and 72 h after XF inoculation were treated in DAB buffer under light for 8 h. Following absorption of the DAB solution, leaves were destained in the solution (absolute ethyl alcohol: acetic acid, 1:1 v/v) and then immersed in chloral hydrate until they became clear. Only infection sites in which substomatal vesicles had formed were considered successful penetrations. A total of 60 infection sites per treatment were observed with Bright-field microscopy, and the ROS area was calculated using CellSens Entry software. The infestation rate of Pst was calculated as the number of the infected sites (20) divided by the total number of stomata evaluated (N) according to the method with minor modifications (Tang et al. 2015a). The assay was repeated three times.

Histological observation of fungal growth

Sampled leaves were fixed and stained as previously described (Tang et al. 2015b) until the samples became transparent. After incubation in 2.0 mL of 1 M KOH in a boiling water bath for 30 min, the leaves were rinsed three times with 50 mM Tris–HCl (pH 7.4) for 15 min and incubated in WGA Alexa488 solution (cat. no. W11261, Thermo Fisher Scientific). Fifty infection sites were observed with an Olympus BX-51 microscope. The infected areas, hyphal lengths, and haustorial mother cells were observed and used for CellSens Entry software (version: V1.7) calculations.

Scanning electron microscopy

Sampled leaves were treated for SEM examination as described by (Li et al. 2013). Pst growth in leaves was observed using a Nano SEM-450. Leaves were collected and treated for SEM as described. The observation of XF growth on leaves was performed following the above description.

RNA extraction and reverse transcription-quantitative PCR (RT-qPCR) analysis

Total RNA of the harvested leaves was extracted with Trizol and converted to cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser following the manufacturer's protocols. Biomass quantification was performed as described by Xu et al. (2019), and the biomass of wheat and Pst was determined by the internal reference genes TaEF (wheat elongation factor 1 alpha) and PstEF (rust elongation factor 1). In addition, the expression of SA- (PR1, PR2, and PAL) and JA/ET-related genes (PR3, PR4, and PR9) in leaves were determined, and the TaEF gene was used as the internal control gene. All primer sequences are shown in Additional file 2: Table S4. RT-qPCR was performed using UltraSYBR Mixture (CWBIO, China). Each treatment contained three independent biological replicates, and each replicate contained three technical replicates. Relative transcript levels were calculated using the 2^–ΔΔCT method (Livak and Schmittgen 2001).

Preparation of crude extracts

Four solvents, PE, DCM, EA, and NB, were used sequentially in a polar gradient approach to define the polarity range of active substances in XF FL (Chen et al. 2021). Specifically, we extracted 100 mL of FL with an equivalent volume of PE overnight. After incubation, the solution was separated into two layers, and the target phase was collected and concentrated with a vacuum concentrator. The remaining phase was extracted overnight with an equivalent volume of DCM. In subsequent procedures, we recovered the PE, DCM, EA, NB, and R extracts. All extracts were dissolved in 0.5% methanol to 50 mg/L and used for antifungal activity assay.

XF FL was extracted with an equal volume of EA overnight. Following full extraction, the mixture was separated into two layers. The EA upper layer was then evaporated using rotary evaporator to yield the EAP. The lower aqueous solution was collected and dried to obtain NEAP. These fractions were dissolved in 100 mL of 0.5% methanol, and stored at 4°C. In addition, protein was removed by incubating 5 mL of EAP and NEAP for 1 h at 37°C with 25 μl of proE K (10 mg/mL, already pre-treated at 58°C for 2 h).

Data processing

All data are presented as mean values ± standard deviation (SD). Data were subjected to analysis of variance (ANOVA) followed by Duncan’s multiple range tests using SPSS11.0 (SPSS Inc., Chicago, IL, USA).