Soil Inoculated with Streptomyces rochei D74 Invokes the Defense Mechanism of Helianthus annuus Against Orobanche cumana

Abstract

Orobanche cumana Wallr. is a root parasitic plant that causes considerable yield losses of up to 50% in sunflower Helianthus annuus plantations. The holoparasite fulfills its entire demand for water, minerals, and organic nutrients from the host’s vascular system. Agronomic practices alone are not effective in controlling this pest. This study investigated the mechanism of a verified plant growth-promoting strain, Streptomyces rochei D74, on the inhibition of the parasitism of O. cumana in a co-culture experiment. We conducted potted and sterile co-culture experiments using sunflower, O. cumana, and S. rochei D74. Our results suggest that the inoculated bacteria invoked the sunflower systemic resistance (SAR and ISR) by increasing the activity of resistance-related enzymes (SOD, POD, PPO, and PAL), the gene expression of systemic resistance marker genes (PR-1 and NPR1), ethylene synthesis genes (HACS. 1 and ACCO1), and JA synthesis genes (pin2 and lox). The expression levels of ISR marker genes (lox, HACS. 1, ACCO1, and pin2) increased by 1.66–7.91-fold in the seedling stage. Simultaneously, S. rochei D74 formed a protective layer on the sunflower root surface, preventing O. cumana from connecting to the vascular system of the sunflower roots. In addition, S. rochei D74 reduced 5DS synthesis of the strigol precursor substance, resulting in a reduction in O. cumana germination. These results demonstrated that the S. rochei D74 strain improved systemic resistance and decreased seed germination to prevent O. cumana parasitism.

1. Introduction

Due to the partial or complete loss of photosynthesis, a parasitic plant invades either the stem or roots of a host plant and acquires nutrients from the host to meet the requirements of its life cycle. As a result, growth retardation often occurs in the host. About 1% of angiosperm species (approximately 4000 species) have been identified as parasitic plants, which widely exist in the world [1,2]. Some parasitic plants infect crop plants and have been a threat to crop yield [3]. The parasitic plant Orobanche cumana Wallr. (1825) (Lamiales: Orobanchaceae), which specifically parasitizes sunflowers, Helianthus annuus, develops a specialized organ called the haustorium to penetrate into the host roots and acquire nutrients, such as carbohydrates, amino acids, organic acids, and mineral salts [4,5]. In particular, a parasitic O. cumana plant produces a large number of tiny and inconspicuous seeds that can be readily dispersed into the environment, thereby contaminating new planting fields or threatening subsequent farming rotations. Therefore, O. cumana, like many other Orobanche species, is listed in the phytosanitary regulations of most countries [6,7]. Orobanche disease outbreaks have occurred worldwide (Figure S1) and led to dramatic economic losses [8]. For example, O. crenata caused yield losses of 50% in Malta and 30% in Egypt, respectively [9]. O. cumana caused yield losses of 60% in Greece and 20–50% in China, respectively [10]. In Spain, 40,000 hectares of contaminated sunflower farmland were abandoned due to the devastating O. cumana parasitism [10]. Strategies were applied to limit the O. cumana parasitism in agricultural practices, such as crop rotation with non-host plants and the application of chemical-based pesticides [11]. However, these strategies are either not sufficient or not environmentally friendly [12]. While researchers have developed resistant O. cumana parasitized sunflower varieties, such resistance could be circumvented by the appearance of new Orobanche isolates [13,14].

The mechanisms of resistance to parasitic plants have been less studied than those against other pathogens or pests. Plants have an innate immune system that triggers defense against numerous potential pathogens through pathogen-associated molecular patterns (PAMPs). The resistance of host plants to parasitic weeds is similar to their resistance to pathogens. This hypothesis is also supported by the fact that tomato responds to a small peptide factor occurring in Cuscuta spp. with immune responses typically activated after the perception of microbe-associated molecular patterns (MAMPs) [15]. Complex signaling pathways related to systemic resistance, often involving the salicylic acid (SA)-dependent systemic acquired resistance (SAR) pathway and jasmonic acid (JA) and/or ethylene-dependent induced systemic resistance (ISR) responses, lead to the activation of suites of defense genes, which are determined by the host–parasite interaction [16]. During the establishment of sunflower resistance to O. cumana, Arabidopsis thaliana resistance to Phelipanche ramosa, and sorghum and rice resistance to Striga hermonthica (Del.) Benth, SA also induces the expression of genes encoding pathogenesis-related proteins (PRs) [17,18,19].

S. rochei D74 is a plant growth-promoting microorganism and has been widely used in plant sunflower cultivation to promote plant growth [20]. In our farming practices on an O. cumana-contaminated farmland, we unexpectedly observed a dramatic inhibition of O. cumana parasitism on sunflower plants after applying S. rochei D74 strains: almost 0% parasitism occurred in the roots of sunflowers (Figure S2). However, the current research remains limited to superficial phenomena, with insufficient investigation into how applied methods affect the parasitism of O. cumana. Therefore, this study employs laboratory experiments to elucidate how S. rochei D74 enhances plant immune systems and reshapes rhizosphere microbial communities, thereby effectively suppressing O. cumana parasitism. The elucidation of this mechanism provides a theoretical foundation for the future management of parasitic plants.

2. Materials and Methods

2.1. Plant, Weeds, and Strain

Sunflower seeds of the Orobanche cumana-susceptible species (H. annuus) variety ‘363’ were purchased from the Sanrui Agritec Co., Ltd. (Bayannur City, Inner Mongolia Autonomous Region, China). O. cumana, as a weed, was collected from a parasitized sunflower farm in Bayannur City. The actinomycete S. rochei D74 (GenBank Accession No. KJ145878) was isolated from healthy danshen (Salvia Miltiorrhiza Bge.) soil in Shaanxi Province. The S. rochei D74 strain was screened, identified, and preserved by our team at the Laboratory of Microbial Resources, College of Natural Resources and Environment, Northwest A&F University (Yangling, Shaanxi Province, China). A spore powder of S. rochei D74 was prepared by solid-state fermentation, and the resulting strain had a total viable count of 5 × 10¹⁰ colony-forming units/g.

2.2. Sunflower, O. cumana, and S. rochei D74 Co-Culture Experiments

Seeds of O. cumana were surface-sterilized with 1% sodium hypochlorite solution [21]. S. rochei D74 was used in seed germination tests. Cell-free fermentation broth was obtained by high-speed centrifugation and suction filtration after culturing in Gause’s No. 1 medium for 7 days. The sunflower seeds were surface-sterilized and placed in one cavity (size 5 cm × 5 cm × 15 cm, soil + perlite substrate at ratio 2:1), which was filled with 20 mL of sterile distilled water and incubated in an artificial climate chamber (28 °C, light (12,000 lux) and dark incubation times of 12 h:12 h)). After the plants had developed two true leaves (7–10 days), the seeds with uniform growth were uprooted from the soil, and the roots were washed with sterile water and set aside.

On a clean bench, the sunflower root was laid flat on a 9 cm diameter glass fiber filter paper evenly sprinkled with 15 mg of surface-sterilized O. cumana weeds, and another glass fiber filter paper was placed on top to create a sandwich-like shape. Two pieces of ordinary filter paper of the same size were then attached to each side of the sandwich model, and four layers of filter paper were stitched together with a stapler so that the seeds and sunflower roots would not become loose. To keep seedling leaves on the outside, the co-culture filter paper was placed in a 9 cm plastic bag, and 20 mL of Hoagland nutrient solution [22] was added, followed by a 2-day replenishment of the nutrient solution. A tinfoil-wrapped opaque carton was used to keep the roots from receiving direct light. Observations on O. cumana parasitism were conducted after 60 days of incubation in an artificial climate chamber (28 °C, 12 h light/12 h dark, Figure S3a).

For observation of the S. rochei D74 colonization site in sunflower roots, we used S. rochei D74 tagged with green fluorescent protein [23]. Surface-sterilized sunflower seeds were placed in a co-culture filter paper model with O. cumana seeds added beforehand, and then 20 mL of Hoagland nutrient solution was added, followed by 500 μL of S. rochei D74-GFP culture solution at 0 and 30 days, respectively. After 60 days of above-described incubation, O. cumana parasitism was observed in sunflower roots using confocal laser scanning microscopy (Leica Microsystems, Heidelberg, Germany).

2.3. Pot Experiments Setup

Pot experiments were conducted in the Mobile Water Control Crop Shed, Research Institute of Arid Area Water-saving Agriculture, Northwest A&F University, China. The soil comprised arable soil from farmland in Yangling, Shaanxi Province (108.075° E, 34.282° N). The pots were almost cylindrical, with a diameter of 24.5 cm and height of 27.5 cm. Each pot contained 10 kg of mixed soil from local farmland. Soil physicochemical properties are Lou soil; pH 8.5; total potassium, 17.19 g/kg; total nitrogen, 0.52 g/kg; and total phosphorus, 0.72 g/kg. One kg of mixed soil contained 5% organic fertilizer (Organic matter ≥ 40%, NPK content > 5%), 0.43 g urea, and 0.15 g single superphosphate. The S. rochei D74 experiments were established in spring of 2019 with four different treatments: (1) C1 (no S. rochei D74; no O. cumana seeds), (2) C2 (added S. rochei D74, 1.5 g/kg soil; no O. cumana seeds), (3) T1 (no S. rochei D74; added O. cumana seeds, 3.4 mg/kg soil), (4) T2 (added S. rochei D74, 5 g/kg soil; added O. cumana seeds, 3.4 mg/kg soil). Each treatment had 46 replicate pots, and six of the pots were not planted and served as the corresponding plant-free control (such as NPC1, NPC2, NPT1, and NPT2). The remaining 40 pots were planted with three sunflower seeds and one was retained from the growth of one real leaf stage. Weeding and irrigation were performed regularly to ensure normal plant growth (Figure S3b).

To determine the colonization site of S. rochei D74, for pot experiments, we collected healthy sunflowers and O. cumana. In order to separate the epiphytic and endophytic actinomycetes from each tissue, the top, middle, and roots of O. cumana, as well as the leaves, stems, and roots of sunflower, were each ground separately [24,25]. The surface of Gause’s No. 1 medium was coated with tissue grinds of 10⁻¹ and 10⁻² concentrations for the identification of actinomycetes. We also collected soil from the rhizosphere of sunflower roots to isolate and culture actinomycetes.

2.4. Plant and Soil Sampling

At plant seedling stage (SS), budding stage (BS), flowering stage (FS), and maturity stage (MS), about nine individual sunflower plants were randomly selected from each treatment. Two healthy leaves at upper position of each plant were washed with distilled water, clipped, and immediately placed in an ice bag. The leaves were chopped and dispensed into sterile test tubes on an ultra-clean table, immediately wrapped with tin foil, placed in liquid nitrogen, and brought to the laboratory, where they were kept in a freezer at −80 °C until needed. Later on, they were used for the quantitative analysis of gene expression, defense enzyme activities, and endogenous hormones. At each treatment and development stage, the sunflower plants were harvested, and the height and dry weight of the host, as well as the aboveground number, underground number, and dry weight of O. cumana, were recorded. The rhizosphere soil was collected and immediately placed in an ice bag. In total, we collected 126 samples at sunflower seedling, budding, flowering, and mature stages for microbial community analysis, which included 96 samples from six compartments, 24 samples of plant-free control, and six farmland soil samples (BK).

2.5. Measurement of Endogenous Hormone Levels and Resistance-Related Gene Expression in Sunflower Leaves

Apical leaves of three plants were measured as a mixed sample. Phytohormone contents of salicylic acid (SA), jasmonic acid (JA), and strigolactones (SLs) were detected by MetWare biotechnology Co., Ltd. (www.metware.cn/ accessed on 13 April 2021) based on the AB Sciex QTRAP 6500 LC-MS/MS platform. Plant materials (50 mg fresh weight) were frozen in liquid nitrogen, ground into powder, and extracted with 1 mL methanol/water/formic acid (15:4:1, V/V/V). The combined extracts were evaporated to dryness under nitrogen gas stream, reconstituted in 100 μL 80% methanol (V/V), and filtered through 0.22 μm filter for further LC–MS analysis [26,27]. Detection details are in the Supplementary Materials Text S1 and Table S1. JA compounds include jasmonic acid (JA) and jasmonoyl-L-isoleucine (JA-ILE). SA compounds include salicylic acid and salicylic acid 2-O-β-Glucoside (SAG). Strigolactone compounds are mainly 5-Deoxystrigol (5DS).

Total RNA was extracted from sunflower leaves using the EasyPure^® Plant RNA kit (TransGen Biotech Co., Ltd., Beijing, China). The RNA extract concentration and purity were measured by an Epoch™ microplate spectrophotometer (BioTek, Winooski, VT, USA) and were reverse-transcribed by EasyScript^® First-Strand cDNA Synthesis SuperMix (TransGen Biotech Co., Ltd., Beijing, China). RT-qPCR was performed using the PerfectStart^® Green qPCR SuperMix (TransGen Biotech Co., Ltd., Beijing, China). Then, these DNA samples were used for real-time fluorescent quantitative PCR (RT-qPCR) on a QuantStudio 7 real-time PCR system (ThermoFisher Scientific, San Jose, CA, USA). The PCR reaction (20 μL) consisted of the following: 10 μL of 2 × PerfectStart^® Green qPCR SuperMix, 1.0 μL of the DNA template, 0.4 μL each of the forward and reverse primers (10 μM), 0.4 μL passive reference dye (50×), and 7.8 μL of nuclease-free H₂O. The reaction conditions were as follows: pre-denaturation at 94 °C for 30 s, followed by 40 cycles of denaturation at 94 °C for 5 s, and annealing at 60 °C for 30 s.

The EF-1a gene was selected as the reference gene when analyzing a target gene. The expression of systemic resistance marker genes (PR-1 and NPR1), ethylene synthesis genes (HACS. 1 and ACCO1), and JA synthesis genes (pin2 and lox). The nucleotide sequences of primers are listed in Table S2 [14,28,29,30,31]. Three technical replicates were set up for each sample. The relative expression level of the targeted genes was calculated using the 2^−ΔΔCT method. Data analysis was carried out using GraphPad Prism 9 statistics software [32].

2.6. 16S rRNA Gene Sequencing and Bioinformatic Analysis

Total genomic DNA was extracted from the soil samples using a FastDNA SPIN Kit for Soil (MP Biochemicals, Solon, OH, USA) according to the manufacturer’s protocol. The prepared DNA samples were submitted to Novogene Co., Ltd. (Tianjin, China) for library preparation and sequencing. Polymerase chain reaction was performed with the 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 907R (5′-CCGTCAATTCCTTTGAGTTT-3′) primer pair [33], which amplified variable regions 4 and 5 in the 16S rRNA gene. 16S rRNA amplicon sequencing was generated on an Illumina NovaSeq platform and 250 bp paired-end reads [34,35,36,37]. Using USEARCH, we relabeled sequencing names, removed barcodes and primers, filtered low-quality reads, and identified non-redundant sequences [34,37]. Unique reads were clustered into operational taxonomic units (OTUs) with ≥97% similarity. Representative sequences were selected with UPARSE (version 7.1) [38] and aligned with the SILVA database (release 132) [39] using the Mothur algorithm [40] to annotate taxonomic information. In order to study the phylogenetic relationships among different OTUs and the different dominant species in each of the samples (groups), multiple sequence alignment was conducted using MUSCLE software (version 3.8.31) [41]. The α-diversity index, distance matrix calculation, and barplot production were obtained using the vegan [42] and ggplot [43] packages in R (version 4.0.0, https://www.r-project.org/ accessed on 20 October 2022). Constrained PCoA (CPCoA) plots were obtained using the ImageGP web server (http://www.ehbio.com/ImageGP/ accessed on 10 November 2022).

2.7. Statistical Analysis

The statistical analyses of the data collected from the field and pot experiments were conducted using GraphPad Prism 9 statistics software [32] and Excel 2020 (Microsoft Corp., Redmond, WA, USA). The means of the response variables—namely, plant biomass, culturable microbial counts, enzyme activities, hormone levels, gene expression, alpha diversity, and abundance of culture-independent bacterial communities—were compared between the S. rochei D74 and control treatments by two-way ANOVA test.

3. Results

3.1. The S. rochei D74 Strain Inhibited O. cumana Parasite and Facilitated Sunflower Growth

Through a sunflower–O. cumana co-culture experiment, the addition of S. rochei D74 bacterial solution in a sterile environment significantly reduced the parasitism of O. cumana, and almost no O. cumana seeds parasitized sunflower roots (Figure 1a). In the control, O. cumana seeds successfully parasitized the sunflower root system and formed tubercles (Figure 1b).

In the pot experiment, the amount of parasitism of each sunflower plant was counted, including the aboveground emerged part and the belowground part, which were able to parasitize the hosting sunflower plants. In comparison with the control, the number of parasites was reduced by 29.4% after adding S. rochei D74 to BS (budding stage, Figure 2a). As the plant developed into the FS (flowering stage), parasitic and emergent O. cumana showed rapid growth, while as the plant was treated with the S. rochei D74, fewer O. cumana developed. The S. rochei D74 strain drastically decreased by 29.9% the parasitism number of O. cumana during the host’s MS (maturity stage, Figure 2a). The dry weight of the sunflower was measured four times in pot experiments, and the results showed that the addition of the S. rochei D74 strain significantly increased by 30.4% (C2 compared with C1) and 22.5% (T2 compared with T1) at the FS, respectively (Figure 2b). Regardless of whether O. cumana seeds were present in the soil (T1, T2) or not (C1, no S. rochei D74, no O. cumana seeds; C2, added S. rochei D74, 1.5 g/kg soil, no O. cumana seeds), sunflower dry matter mass peaked during the flowering period, which is also a period of rapid root development (Figure 2b). Sunflower root dry weight can be enhanced by adding the S. rochei D74 strain (without O. cumana seeds, C1 vs. C2: 21.92%; with O. cumana seeds, T1 vs. T2: 120.17% (Figure 2b)) at the FS. The root growth and development of sunflowers were delayed in the T1 treatment group because the parasitism caused the nutrients of sunflower roots to move to the O. cumana to satisfy their own development. S. rochei D74, on the one hand, inhibits the parasitism of O. cumana; however, as a plant growth-promoting strain, it supplies nutrients to the host and may promote root development [44]. We verified how the bacterial community changed after adding O. cumana and S. rochei D74.

3.2. The O. cumana and S. rochei D74 Strain Effect on Bacterial Microbiota Across Four Developmental Stages of Sunflower

The raw sequence data reported in this paper have been deposited in the NCBI under accession number BioProject: PRJNA830293. The average sequencing data statistics for each sample are as follows: the number of original PE reads is 94,483, the number of Raw Tags is 93,955, the number of clean tags is 91,332, the number of Effective Tags is 62,633, the base is 23,429,864 nt, the average length is 3,741 nt, the Q30 is 94.46, and the GC content is 57.15% (Table S3).

The rhizosphere bacterial Shannon index was increased at the BS and then decreased over time according to the alpha diversity analysis. As in the C1, C2, and T1 groups, the Shannon index for the host rhizosphere bacterial species increased with bud emergence and decreased with flowering, whereas in the T2 group, the Shannon index increased with seedling emergence and then showed a continuous decline (Figure 3a). Under conditions with bacteria and O. cumana combined, the diversity appeared to be reduced (Figure 3a and Figure S6). The structure of the community differed significantly with and without sunflower plants, according to the constrained principal coordinate analysis (CPCoA) analysis of the microbial community, suggesting that planting sunflowers may cause shifts in bacterial community structure. Without plants, NPC1 (based on C1, but with no planted sunflowers) and NPC2 (based on C2, but with no planted sunflowers) overlapped, and NPT1 (based on T1, but with no planted sunflowers) and NPT2 (based on T2, but with no planted sunflowers) overlapped. Furthermore, the experimental group was evaluated according to the host growth phase, and the findings revealed that the seedling stage (SS) and the budding stage (BS) partly overlapped, as did the flowering stage (FS) and maturity stage (MS) (Figure 3b). These two clusters were well separated in the two distinct regions of CPCo1–CPCo2 (Figure 3c). At this stage, plants change from nutritional to reproductive development. In summary, the microbial communities in the experimental group were divided not by treatment but by sampling period, demonstrating that the varied growing phases of sunflower had a stronger influence on microorganisms than the treatment.

Figure 3. Effect of S. rochei D74 inoculation on soil microbial community composition. (a) Shannon index, CPCoA (b,c) based on Bray–Curtis distance in different experiment groups of treatments and the growth period of sunflower. BK, original farmland soil samples without any treatment; NP, soil of no planted sunflowers. Different lowercase letters indicate significant differences (p < 0.05, Fisher’s LSD test). The rhizosphere microorganisms revealed (Figure 4a) that C1 and C2 communities and T1 and T2 communities were similar without plants, indicating that O. cumana was the main factor influencing the microbial community structure in the absence of plants. The predominant differential microorganisms during the host’s nutritional development were Gemmatimonadaceae, Nitrosomonadaceae, Nitrospiraceae, Burkholderiaceae, and Tepidisphaeraceae. The only bacterial family that significantly differed in the reproductive growth period was Burkholderiaceae, whose relative abundance in T2 was always lower than that in T1 in the budding, flowering, and maturation stages (Figure 4b). The functional prediction of the microbial community using the PICRUSt tool and CPCoA analysis of the KEGG Orthology (KO) pathway showed that the seedling stage overlapped with the budding stage, while the flowering stage overlapped with the mature stage (Table S4). Clustering into two distinct areas indicated that the pre- and post-reproductive functions expressed were distinct (Figure 4c). In the case of the flowering stage alone, functional variation was mainly due to the presence of O. cumana in the soil (Figure S7).

Figure 3. Effect of S. rochei D74 inoculation on soil microbial community composition. (a) Shannon index, CPCoA (b,c) based on Bray–Curtis distance in different experiment groups of treatments and the growth period of sunflower. BK, original farmland soil samples without any treatment; NP, soil of no planted sunflowers. Different lowercase letters indicate significant differences (p < 0.05, Fisher’s LSD test). The rhizosphere microorganisms revealed (Figure 4a) that C1 and C2 communities and T1 and T2 communities were similar without plants, indicating that O. cumana was the main factor influencing the microbial community structure in the absence of plants. The predominant differential microorganisms during the host’s nutritional development were Gemmatimonadaceae, Nitrosomonadaceae, Nitrospiraceae, Burkholderiaceae, and Tepidisphaeraceae. The only bacterial family that significantly differed in the reproductive growth period was Burkholderiaceae, whose relative abundance in T2 was always lower than that in T1 in the budding, flowering, and maturation stages (Figure 4b). The functional prediction of the microbial community using the PICRUSt tool and CPCoA analysis of the KEGG Orthology (KO) pathway showed that the seedling stage overlapped with the budding stage, while the flowering stage overlapped with the mature stage (Table S4). Clustering into two distinct areas indicated that the pre- and post-reproductive functions expressed were distinct (Figure 4c). In the case of the flowering stage alone, functional variation was mainly due to the presence of O. cumana in the soil (Figure S7).

3.3. The S. rochei D74 Strain Putatively Invoked Systemic Resistance of Host to O. cumana

Because the leaves were already yellowed and wilted at maturity, the hormone levels were examined during the first three stages of sunflower growth. Based on the comparative analysis of hormones with concentrations over 100 ng/g, we found that the hormone levels of four JA analogs (JA, JA-ILE) and two SA analogs (SA, SAG) were significantly increased in seedling sunflowers in soil supplemented with the S. rochei D74 strain. In the absence of O. cumana in the environment, the addition of the S. rochei D74 strain increased the hormone content of JA (239.0%), JA-ILE (82.1%), SA (66.8%), and SAG (31.3%), which was significant when there were O. cumana seeds in the environment (Figure 5a, p < 0.05). Plants treated with the S. rochei D74 strain had lower levels of 5-Deoxystrigol (5DS, Figure 5a) hormone production in the presence of O. cumana during the growth period, which is a precursor substance to strigolactones (the pathway is shown in Figure S8).

In the seedling stage, regardless of the presence or absence of O. cumana seeds in the growing soil, the expression levels of SAR marker genes PR-1 and NPR1 were higher than in control plants by 3.41- and 2.89-fold with no added O. cumana seeds and by 3.02- and 7.10-fold with O. cumana seeds added (p < 0.05, Figure 5b). At the budding stage, the ISR marker genes lox, HACS.1, ACCO1, and pin2 were active, and gene expression increased 7.44, 7.91, 7.56, and 4.11-fold in the absence of O. cumana and 3.67, 1.88, 2.48, and 1.66-fold in the presence of O. cumana, respectively. Furthermore, the S. rochei D74 strain stimulated the expression of host ISR-related resistance genes when planted without the addition of O. cumana (C1 vs. C2). Comparing C1 with T1, as well as C2 with T2, the presence of O. cumana decreased the expression of resistance genes in the host sunflower at different times.

Under confocal laser scanning microscopy, it was observed that some S. rochei D74-gfp colonies were attached to the nodule surface of O. cumana and the sunflower root surface (Figure 5c), showing a dense dot-like distribution, while no S. rochei D74 strain colonies were found inside the root tissue in sunflower, O. cumana, and actinomycete co-culture experiments in plastic bags. Simultaneously, in the pot experiment, we isolated and screened several sunflower and O. cumana tissue parts, and the results revealed that the S. rochei D74 strain was cultured in the sunflower root surface and rhizosphere soil, but not in sunflower (inside the root, stalk, and leaf) and O. cumana tissues. This indicates that S. rochei D74 strain colonization occurred on the root surface. The S. rochei D74 strain forms a protective layer on the root surface to reduce the number of sprouts of O. cumana and directly stimulate systemic resistance in the host.

4. Discussion

Microorganisms play an important role in plant growth by resisting the invasion of external pathogens. In this present study, the results showed that the S. rochei D74 strain may promote sunflower growth, mainly in terms of plant height and dry matter accumulation. Strigolactones and sesquiterpene lactones released from the hosts have been found to stimulate the germination of root parasites from the Orobanchaceae family and enable weeds to acquire host specificity [45]. What is the reason, however, that the germinated O. cumana did not effectively connect to the roots? How does the physiology of sunflowers change with the addition of the S. rochei D74 strain? Further experiments must be performed. The S. rochei D74 strain inoculation into the soil causes of the host, such as 5DS. This not only stimulates O. cumana germination but also serves as a precursor compound of strigolactone, which is a stimulant for seed germination. Thus, O. cumana was suppressed at the beginning of its growth.

It is also accompanied by the SA-dependent SAR and the JA-dependent ISR pathway of endogenous hormone production. The SAR provides immune memory to the host for several weeks. The host acquires greater resistance during a second attack by the same pathogen, as well as other pathogens, and can even be transmitted to subsequent generations [46]. Previous research has shown that SA decreases the percentage of stages in which the haustoria succeed in penetrating into the central cylinder of the host. As a result of this, SA significantly reduced the number of established parasites due to inhibited radicle growth and the activation of defense responses in the host root [47]. In addition, when Trifolium pratens was treated with JA and SA, it was more resistant to O. minor. This is because it activates defenses that lead to lignification of the endoderm [47]. The addition of the S. rochei D74 strain invoked systemic resistance in sunflowers by increasing the secretion levels of endogenous hormones JA and SA, which possibly may be responsible for the inhibition of sprouting and parasitism of O. cumana. For resistance-associated enzymatic activity (Table S5), O. cumana invasion is similar to that of pathogenic bacteria in that both triggered PAMP immunity responses in the host. The S. rochei D74 strain enhances defense-related SOD, PPO, and PAL activities. Collectively, these results indicate that the S. rochei D74 strain treatment simultaneously activated both the ISR and SAR resistance pathways in sunflowers. Their activation may lead to the accumulation of callose in the intercellular space [48], effectively preventing the germ tube from reaching the root. The S. rochei D74 colonized the root surface, a niche that improved its efficacy in resisting parasitic infection.

The plant development stage had a greater impact on the microbial communities than O. cumana. The alterations in the community were most noticeable during the flowering period, and the reproductive development of the plant was a watershed moment for the microorganisms. From the flowering stage until maturity, the bacterial community structure tended to stabilize. They were similar among the groups in the maturity stage, which provided us with reliable sample options for future investigations of microbial communities. The plant developmental stage strongly influences the role of plant microbiota. Keystone species perform different ecological functions during various developmental stages. The major role of the rhizosphere microbiota in the host’s reproductive period is the permease protein and ATP-binding protein of the ABC transport system, according to function prediction. Microbial communities change with host growth. The primary differential bacteria were Gemmatimonadaceae, Nitrosomonadaceae, Nitrospiraceae, and Burkholderiaceae, which tended to be balanced as sunflowers developed to maturity (Figure 3). In short, the influence of O. cumana and S. rochei D74 strains on rhizosphere microorganisms was smaller than the effect of the plant phenophase on microorganisms, with the most significant variations occurring during the reproductive phase.

Briefly, the S. rochei D74 strain, as the verified plant growth-promoting strain, may promote the growth of host sunflowers while also increasing the resistance of the sunflower system by increasing the activity of resistance-related genes and enzymes. In contrast, the S. rochei D74 strain, as the protective layer of the sunflower root surface, reduced the synthesis of the host strigol precursor substance 5DS, resulting in a reduction in O. cumana germination. In summary, through a multifaceted intervention, the S. rochei D74 strain inhibited O. cumana parasitism.

5. Conclusions

This comprehensive study systematically analyzed the putative effects induced by the S. rochei D74 strain and the underlying mechanisms against O. cumana parasitism. The inoculation of soil with the S. rochei D74 strain was effective in reducing the parasitic abundance of O. cumana and increasing sunflower biomass. The pot experiments unveiled the plausible underlying mechanisms involved in some candidates, in that S. rochei D74 treatment enhanced defense-related SOD, POD, PPO, and PAL activities and increased jasmonic acid and salicylic acid compound secretion in sunflower leaves. Importantly, we observed a reduced secretion of 5DS when the S. rochei D74 strain was added, which is a prerequisite for SL synthesis to promote seed germination. Moreover, S. rochei D74 treatment upregulated the expression of key genes related to the SAR and ISR pathways (NPR-1, PR-1, lox, HACS.1, ACCO1, and pin2). Finally, applying the S. rochei D74 strain to soil increased the diversity of bacteria and the abundance of potentially beneficial microbes in the rhizosphere of sunflower plants. In addition, the rhizosphere microbial community composition and function of sunflowers dynamically changed with the developmental phase of the host, opening new avenues for more sustainable resistance to sunflower broomrape and parasitic plants in crops.