Research

A potent endophytic fungus Purpureocillium lilacinum YZ1

protects against Fusarium infection in field-grown wheat
Roy Njoroge Kimotho1,2*, Xin Zheng1* , Furong Li1, Yijun Chen1,2 and Xiaofang Li1
1
Centre for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Shijiazhuang, 050021, China; 2University of Chinese Academy of
Sciences, Beijing, 100049, China

Summary
Author for correspondence:
Fusarium diseases pose a severe global threat to major cereal crops, particularly wheat.
Xiaofang Li Existing biocontrol strains against Fusarium diseases are believed to primarily rely on antago-
Email: [email protected]
nistic mechanisms, but not widely used under field conditions.

Here, we report an endophytic fungus, Purpureocillium lilacinum YZ1, that shows promise
Received: 8 November 2023 in combating wheat Fusarium diseases. Under glasshouse conditions, YZ1 inoculation
Accepted: 10 June 2024
increased the survival rate of Fusarium graminearum (Fg)-infected wheat seedlings from 0%
to > 60% at the seedling stage, and reduced spikelet infections by 70.8% during anthesis. In
New Phytologist (2024) field trials, the application of YZ1 resulted in an impressive 89.0% reduction in Fg-susceptible
doi: 10.1111/nph.19935 spikelets.

While a slight antagonistic effect of YZ1 against Fg was observed on plates, the induction of
wheat systemic resistance by YZ1, which is distantly effective, non-specific, and long-lasting,
Key words: biocontrol, Fusarium diseases,
growth-promoting endophyte, induced appeared to be a key contributor to YZ1’s biocontrol capabilities. Utilizing three imaging
systemic resistance, Purpureocillium methods, we confirmed YZ1 as a potent endophyte capable of rapid colonization of wheat
lilacinum, wheat. roots, and systematically spreading to the stem and leaves. Integrating dual RNA-Seq, photo-
synthesis measurements and cell wall visualization supported the link between YZ1’s
growth-promoting abilities and the activation of wheat systemic resistance.

In conclusion, endophytes such as YZ1, which exhibits non-antagonistic mechanisms, hold
significant potential for industrial-scale biocontrol applications.

led to the emergence of Fg variants resistant to them (Chen

Introduction
et al., 2018; Xu et al., 2022). The escalation of pesticide resis-
Fungal infections lead to 10–23% annual loss in global crop pro- tance has become a major concern in recent years, underscoring
duction, despite the widespread use of antifungals (Stukenbrock the pressing need for alternative biocontrol strategies, which are
& Gurr, 2023). Particularly, Fusarium graminearum (Fg) is one currently limited.
of the devastating fungal pathogens world-wide, causing Fusar- In nature, plants associate with a wide variety of microbes, col-
ium crown rot (FCR) and Fusarium head blight (FHB) in small lectively known as the plant microbiome (Legrand et al., 2017).
grain cereals, such as wheat, which hold significant economic These associations benefit the plant by promoting growth as well
importance globally. FCR can infect winter wheat at all growth as suppressing pathogens (Sarkar et al., 2019; Tian et al., 2020).
stages, resulting in yield reductions of up to 43% in Turkey, As fungicide resistance worsens and the availability of fungicides
45% in Iran, and 35% in the United States (Kazan & Gardi- for disease control diminishes, there is a growing interest in har-
ner, 2018; Su et al., 2021). Similarly, wheat FHB has caused agri- nessing plant-associated microbes for pathogen management
cultural losses exceeding $3 billion in the United States and (Mousa et al., 2016). Several bacterial strains, including those
Canada (Ward et al., 2008). Furthermore, FHB produces myco- from the genera of Bacillus (Xu et al., 2020; Cantoro et al.,
toxins that pose a significant threat to both humans and livestock 2021), Pseudomonas (Wang et al., 2015; Bubici et al., 2019), and
(Johns et al., 2022). Recent reports indicate that FHB outbreaks Streptomyces (LeBlanc, 2022), have successfully reduced FHB
are on the rise in Asia, Europe, and South America, posing an incidence and severity in the field. Similarly, positive outcomes
increasing threat to the global food supply (Legrand et al., 2017). have been reported with fungal biocontrol agents (BCAs) such as
Due to the lack of resistant cultivars, chemical fungicides are Cryptococcus spp. (Zhang et al., 2022), Trichoderma (Mondani
commonly used to manage FHB in wheat (Blandino et al., et al., 2021), Simplicillium (Abaya et al., 2021), Clonostachys spp.
2012). Unfortunately, the prolonged use of these fungicides has (Abdellatif et al., 2022), and various other species (Comby
et al., 2017; Rojas et al., 2020; Hao et al., 2021; Noel et al.,
*These authors contributed equally to this work. 2022). Despite more than 70 yr of intensive research and

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promising results, only a few BCAs are currently commercially F. graminearum (Fo609) (Fg), F. oxysporum Schlecht. (0685)
available, with Serenade ASO (Bayer) being used to protect (Fo), and F. pseudoanthophilum (5642) (Fp) were used in this
against different Fusarium species, and Polyversum® specifically study. Fg was propagated at 28°C on PDA. For microconidia
designed for FHB management (Milofsky, 2007). Stringent legis- production of YZ1 and Fg, both strains were grown in their
lation is one of the reasons responsible for the lack of effective respective media without the addition of agar for 5 d at 28°C. A
BCAs (Frederiks & Wesseler, 2019). Moreover, the majority of winter wheat cultivar Jimai 22 was used. This cultivar is known
existing BCAs targeting Fg are believed to operate through antag- to be susceptible to FHB. The seeds were a collection of our cen-
onistic mechanisms involving the production of antibiotic agents, ter. Seeds were surface-sterilized with 70% ethanol for 2 min
parasitism, and nutrient competition (Shah et al., 2018). How- thoroughly, rinsed five times for 1 min each time with sterile dis-
ever, recent studies focusing on the mechanisms employed by dif- tilled water, and incubated in 2% sodium hypochlorite for
ferent BCAs have revealed different strategies used by these BCAs 15 min. The surface-sterilized seeds were placed on wet sterile fil-
to control different plant pathogens, including Fg (Lee et al., ter papers on Petri dishes sealed with micropore tape and kept in
2021; Karuppiah et al., 2022; Pescador et al., 2022). Further- the dark for germination at room temperature. Five-day-old seed-
more, endophytic BCA strains are considered superior candidates lings of similar size were transferred to ½-strength Murashige &
due to their diverse beneficial effects and enhanced environmen- Skoog medium (½MS) before inoculation. The soil used for
tal adaptability under field conditions. A significant challenge all the glasshouse experiments was collected from a field in the
lies in translating the biocontrol capabilities observed in the Luancheng Agroecosystem Experimental Station (37°53 0 N,
laboratory to effective outcomes in the field, often resulting 114°41 0 E) in Luancheng County, Hebei Province, China. The
in reduced efficacy of known BCAs under field conditions field had an extended history of maize and wheat rotation and
(Li et al., 2024). were not exposed to agrochemicals for several years before collec-
In this study, we report a recently discovered endophytic fun- tion. The soil was fluvo-aquic with a total nitrogen of 1.13–
gus, Purpureocillium lilacinum L. YZ1, which demonstrates sig- 1.70 g kg1, a total carbon of 17.03–20.80 g kg1, and a pH of
nificant positive effects on wheat growth and suppression of 7.53–7.95.
Fusarium disease. Through the use of advanced imaging techni-
ques, we observed that YZ1 could colonize wheat plants endo-
Preparation of green fluorescent protein (GFP)
phytically and migrate to aboveground parts without inducing
expressing YZ1
any visible symptoms. Upon establishing endophytic coloniza-
tion, YZ1 triggered jasmonic acid (JA)-independent systemic To generate green fluorescent protein (GFP)-labeled YZ1, we
resistance (ISR) against Fg. Tests showed that the ISR is non- initially prepared YZ1 protoplasts and subsequently transformed
specific, long-lasting, and distantly effective. Further, dual them following modified protocols (Shen et al., 2015; He &
RNA-Seq interactions indicate that YZ1 significantly regulates Ewbank, 2017). The detailed methods and chemicals used in the
wheat transcriptome, which may enhance wheat’s defense via preparation of YZ1 protoplasts can be found in Supporting
growth promotion during Fg infection. This study provides novel Information Methods S1.
insights into the biocontrol effects and mechanisms of a
non-antagonistic endophytic strain against Fusarium diseases and
Preparation of the inoculants of YZ1, GFP-YZ1, and Fg
lays a solid foundation for the development of sustainable
approaches to managing FHB in wheat. Green fluorescent protein expressing YZ1 (pCEV-gfp-g418 vec-
tor) and the wild-type (WT) YZ1 were prepared as described
above and diluted with sterile distilled water to a final concentra-
Materials and Methods
tion of 2.5 OD600 units (c. 2.5 9 105 hyphal fragments ml1).
Detailed methods for the preparation of Fg inoculum can be
Growth media and microbial cultivation conditions
found in Methods S1.
One liter of Martins media for culturing Purpureocillium lilaci-
num L. YZ1 contained 10 g glucose, 5 g peptone, 1 g KH2PO4,
Beneficial effects of YZ1 tested in pot-culture experiments
0.5 MgSO47H2O, and 15 g Agar for solid media (Yuan
et al., 1994). For culturing Fusarium graminearum S. (Fg), potato To investigate the beneficial effects of YZ1 on wheat, two
dextrose agar (PDA) was prepared by dissolving 46 g of Coolaber glasshouse experiments were conducted using autoclaved and
Potato Dextrose Agar (Cat no. Pm0520, Shanghai, China) in 1 l non-autoclaved soil. For the sterilized soil experiments, the soil
of ultra-pure water (Liu et al., 2021). All media were autoclaved collected from the field underwent two rounds of autoclaving.
at 121°C for 15 min. Autoclaving the soil helped eliminate potential interference from
existing Fusarium species in the soil, particularly considering the
sampling site’s Fusarium-affected nature. However, no observable
Strains, plants, and soil source
negative impacts on soil structure or nutrient status resulting
YZ1 was isolated from the soil in Luancheng County, Hebei Pro- from autoclaving were noted (Li et al., 2023). The experimental
vince, China. This strain belongs to the mycelial fungus genus treatments included YZ1 only, Fg only, control (mock inocu-
Purpureocillium (Zheng et al., 2021). Local virulent strains of lated), and YZ1 & Fg, which were applied at the time of sowing

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in pots (19 9 12 cm pots, 550 g of soil each). The application

Split-root experiment
of Fg was done using a modified inoculation method (Liu
et al., 2021). Detailed methods for the pot-culture experiments To perform the induced resistance (IR) test, a split-root system
can be found in Methods S1. This experiment was repeated three was used in order to create a physical separation between YZ1
times independently. and Fg during their interaction with wheat roots. Wheat seedlings
The YZ1 treatment showed very similar results in growth pro- were pre-inoculated with YZ1 on one side of the split-root sys-
motion and biocontrol effect in both sterile and non-sterile soil tem, and after 5d, the other side was inoculated with Fg. For the
environments. Consequently, only the results from the sterile Fg treatment, Fg was inoculated on both sides of the split-root
soil treatment were presented here. system. All pots were arranged in a completely randomized
design in the glasshouse. Leaf samples were collected at 4, 24,
and 48 h and stored at 80°C for RNA extraction to assess the
Disease rating of Fg infection under glasshouse conditions
systemic effects of YZ1 on Fg infection. Quantitative PCR was
A conidial suspension was prepared from a 6-d culture of the Fg performed to determine the gene expression dynamics of eight
strain Fo609, which was cultivated in potato dextrose medium. selected immune response genes, which serve as marker genes for
The suspension was filtered through six layers of lens cleaning SA, abscisic acid (ABA), and JA signaling pathways (Table S1).
paper and mixed with Tween 20 (0.01%). Between 4 and 6 d
after full spikelet emergence (Feekes 10.5) (Large, 1954), 10 ll
Quantitative PCR for quantifying wheat genes
of the conidial suspension was diluted to a final concentration of
1 9 105 and inoculated on the fourth spikelet from the base Quantitative PCR was done to track the dynamic response of
using a micropipette. The plants were then kept in a glasshouse wheat defense genes to YZ1 colonization. Methods for RNA
at 26°C with a relative humidity of 90% for 96 h. Fourteen days extraction and qPCR can be found in Methods S1, and all pri-
post inoculation (dpi), symptomatic spikes were examined and mers can be found in Table S1.
photographed. Subsequently, the infection rate of 30 individual
spikes from both YZ1-inoculated and non-inoculated plants was
Microscopy of YZ1’s endophytic colonization
used for statistical analysis. The infected spikelet percentage
was evaluated by the following formula: infected spikelet % = To observe the growth of YZ1 in wheat roots by confocal micro-
(number of symptomatic spikelet’s 9 the total number of spike- scopy, samples were taken from the in vitro experiment between
let’s/100) (Huang et al., 2020). 1 and 28 d. Root sections were washed with phosphate buffer
solution for 10 min and observed for GFP fluorescence using the
488-nm line of the 25-mW Argon ion laser. For the microscopic
Antagonistic activity of YZ1 against the pathogenic
observation of YZ1 colonization using fluorescent dyes and scan-
Fusarium species
ning electron microscopy (SEM), detailed methods can be found
The in vitro antagonistic activities of YZ1 against Fg (Fo609), Fp in Methods S1. All experiments were repeated four times inde-
(5642), and Fo (0685) were evaluated using dual culture assays pendently.
on PDA media. PDA was favored for this dual assay since it pro- Microscopy was performed using tissues from a minimum of
motes growth of different plant pathogenic Fusarium species six plants per treatment, ensuring sterile conditions with only
(Arie, 2019). For the assays, mycelial plugs (0.5 cm in diameter) two organisms present (YZ1 and wheat). Five biological repli-
were collected on the edge of 15-d-old YZ1 cultures grown on cates were derived from four technical repetitions. The experi-
Martins media, or 10-d-old Fusarium cultures grown on PDA ments were replicated in five independent inoculation trials. In
media, and then transferred to plates containing fresh Martins the case of non-inoculated control plants, no fungal hyphae were
media. A plug of YZ1 was inoculated at equidistant points 2 cm detected.
from the edge of the Petri dish, followed by the placement of a
Fg plug 3 cm away from the YZ1 mycelium disk. The dual cul-
Re-isolation of YZ1
ture plates were incubated in the dark at 28°C. The antagonistic
effect was observed after 7 d by measuring the inhibition zones. To analyze the systematic colonization of YZ1 in aboveground
These experiments were repeated three times. wheat tissues, three distinct experiments were conducted. Initi-
ally, surface-sterilized seeds were cultivated in sterile soil in a
glasshouse for 45 d. Detailed methods can be found in Methods
Biocontrol activity of YZ1 secondary metabolites against Fg
S1. This experiment was repeated three times independently.
Based on the results we obtained in the in vitro antagonistic assay
between YZ1 and Fg, we decided to test the effects of YZ1 meta-
Photosynthesis rate and Chl content measurements
bolites on Fg. To obtain the metabolites, batch culture fermenta-
tion of YZ1 was done. Detailed methods for the extraction and The effect of YZ1 inoculation on wheat photosynthesis and rele-
biocontrol tests of YZ1 secondary metabolites can be found in vant parameters were determined. Detailed methods can be
Methods S1. found in Methods S1.

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Evaluating FHB under glasshouse and field conditions Results

The glasshouse infection assay of Fg conidia on flowering wheat
YZ1 promotes wheat growth and increases yield under
spikes was performed as previously described (Tian et al., 2020).
glasshouse conditions
To further assess the efficacy of YZ1 in enhancing wheat resis-
tance against FHB in field conditions, a field trial was conducted The plants were grown to maturity in the glasshouse. We found
in Shijiazhuang City, Hebei Province, China, in 2023. FHB that YZ1-treated plants grew robustly and more vigorously than
resistance was assessed in the third week post-Fg inoculation fol- non-treated plants throughout the growth period (Figs 1a–c, S1).
lowing China’s national standard (NY/T 2954-2016). Detailed At the seedling stage, the average height of plants treated with
methods can be found in Methods S1. YZ1 was 38.9  2.2 cm, significantly greater than that of the
non-treated plants (30.4  2.7 cm) (P < 0.0001) (Fig. 1a,d). At
the booting stage, the average height of inoculated plants reached
Dual RNA-Seq and bioinformatics analyses
57.6  3.4 cm, significantly higher than the non-treated plants
In the in vitro RNA-Seq experiment, each treatment consisted of (49.0  3.6 cm) (P < 0.0001) (Fig. 1b,e). At the anthesis stage,
four individual glass jars, each containing four seedlings. The seed- the mean height of plants treated with YZ1 was
lings were initially germinated on ½MS media. After germination, 62.74  5.3 cm, again surpassing the height of the untreated
the seedlings were carefully removed from the media, inoculated plants (60.7  5 cm) (P < 0.0045) (Fig. 1c,f). Similar trends
with either YZ1, Fg, or a combination of YZ1 and Fg, and then were observed for flag leaf length and width, spike length, and
transferred to fresh ½MS media. The control plants were inocu- stem diameter (Fig. 1g–j).
lated with sterile water. The plants were maintained in a growth YZ1-treated plants also had increased tillering and more robust
chamber with light intensity of 120 lmol m2 s1 and a tillers than the non-treated control plants (Fig. 1l,m). The
16 h : 8 h, 23°C : 19°C, day : night. Roots were harvested at 48 1000-kernel weight, a key indicator of wheat yield, was signifi-
h post inoculation (hpi) and washed thoroughly four times to elimi- cantly higher in YZ1-treated plants than in non-treated plants in
nate extraradical hyphae. The removal of fungal hyphae from the both Trial 1 (37.6  08 g) and Trial 2 (31.8  0.6 g)
root surface was confirmed using light microscopy. Root samples (Fig. 1n).
from the same treatment were combined and promptly snap-frozen
in liquid nitrogen for RNA extraction, which was later used for
YZ1 protects against Fusarium diseases in glasshouse and in
RNA-Seq analysis and quantitative reverse transcription polymerase
field-grown wheat
chain reaction. Experiments were carried out once with eight biolo-
gical replicates. Detailed methods for RNA-Seq and relevant bioin- Symptom analysis revealed that Fg-infected plants exhibited sig-
formatics analyses can be found in Methods S1. nificant FCR symptoms (Fig. 2a), while plants treated with YZ1
and later infected with Fg showed minimal to no symptoms after
15 d (Fig. 2a). Disease severity was calculated as the FCR lesion
Plant hormone assay
length on the coleoptiles, relative to the seedling height (Mori-
Six frozen leaf replicates from the plants grown in the glasshouse mura et al., 2020). Inoculation with YZ1 resulted in significantly
for 15 d were used for hormone analysis. In summary, 100 mg reduced lesions, which were less than 0.35 cm long compared
of leaf samples was finely ground using a pestle and mortar in with the Fg treatment with lesions > 1.75 cm (Fig. 2b). Further-
liquid nitrogen. Subsequently, ABA and JA hormones were more, comparison of FCR disease severity showed that the sever-
extracted and analyzed as previously described (Wang ity was significantly lower in the YZ1 + Fg treatment (1.32%)
et al., 2020). These experiments were performed once with a than in the Fg treatment (13.91%). Overall, inoculation with
minimum of three biological replicates. YZ1 during sowing led to significant growth promotion and pro-
tection against Fg (Fig. 2c). Belowground biomass and root
length were not increased by YZ1 inoculation compared with the
Observations of cell wall thickening
controls (Fig. 2d,e). However, in the YZ1 + Fg treatments, both
Given the significant upregulation of photosynthesis genes root biomass and root lengths were significantly increased com-
observed in the transcriptome results, we hypothesized that the pared with the Fg-alone treatment (Fig. 2d). The aboveground
saccharides produced may contribute to root cell wall thickening. plant biomass was significantly increased by 123.8% by YZ1
Therefore, we conducted experiments to test this hypothesis. inoculation (Fig. 2f). The stem diameter at the base was substan-
Detailed methods for cell wall observations can be found in tially larger at the same time point of 17th day (Fig. 2g). The
Methods S1. This experiment was repeated five independent height of YZ1-inoculated plants was significantly greater
times. throughout the entire time line, from seed emergence to 17 d
(Fig. 2h).
Plant survival rates analyzed by the Kaplan–Meier survival
Statistical analysis
curves showed higher significance in the YZ1-inoculated group
Detailed methods for statistical analysis can be found in than in the non-inoculated when exposed to Fg infections
Methods S1. (Fig. 2i). Survival rates were statistically analyzed using the

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Fig. 1 YZ1 inoculation promotes wheat growth. (a–c) Representative images of side-by-side comparison of YZ1 inoculated and non-inoculated plants at
the seedling (a), bolting stage (b), and anthesis stage (c). (d–f) Height of YZ1-inoculated and non-inoculated plants at the seedling (d), bolting (e), and
anthesis stages (f). Standard error represented by error bars based on five independent biological replicates using 60 plants in each treatment. (g) Wheat
flag leaf length at the anthesis stage (n = 40). (h) Wheat flag leaf width at the anthesis stage (n = 40). (i) Wheat spike length at anthesis stage (n = 40).
(j) Stem diameter of wheat plants at anthesis stage (n = 40). (k) Wheat spike number between YZ1-treated and non-treated plants (n = 40). (l)
Representative image of wheat heads of YZ1-treated and non-treated plants at the anthesis stage in the glasshouse. (m) No. of tillers at the anthesis stage
between YZ1-treated and non-treated control plants (n = 40). (n) The weight of 1000 grains of YZ1-treated and non-treated plants following two
glasshouse trials (n = 50). Different letters indicate significant difference (P < 0.05, Student’s t-test).

Gehan–Wilcoxon test (G–W). A significant difference in mortal- increasing the amount of YZ1 inoculum (Fig. 2i). qPCR quanti-
ity rates emerged between the treatment groups: YZ1 + Fg (105) fication showed a significant decrease in Fg biomass after YZ1
showed a lower survival rate than Fg (105) with a G–W statistic inoculation in both the root and the stem tissues (Fig. 2j). Con-
of 3.083 and a P-value < 0.001. Similarly, when comparing versely, YZ1 load increased significantly after Fg infection in both
YZ1 + Fg (109) with Fg (109), the difference was also statistically the roots and stem tissues (Fig. 2k).
significant (G–W – 3.781, P < 0.001). Interestingly, the protec- FHB symptoms appeared at 17 dpi and spread to the adjacent
tive effect of YZ1 against Fg infection persisted even when spikelets. However, in YZ1-treated plants, the spread of these
the concentrations of the Fg inoculum was increased without symptoms was significantly less (Fig. 3a,b). At 17 dpi, the

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Fig. 2 Effects of YZ1 inoculation and Fusarium graminearum (Fg) infection on wheat. (a) A visual representation of wheat coleoptiles of Fg-infected and
YZ1 + Fg-inoculated plants. Fg-infected coleoptiles had visible brown discoloration while the coleoptiles of seedlings inoculated with YZ1 and infected with
Fg had a white to a light green color. (b) Wheat coleoptile lesion length measurements. Data are mean  SD (n = 15). Different letters indicate
significance (P < 0.05, Student’s t-test). (c) Wheat seedling phenotypes 17 d after treatments. (d) Shoot biomass accumulation at 17 d after treatment
application. Data are mean  SD (n = 40). Different letters indicate significance (P < 0.05, One-way ANOVA). (e) Root length at 17 d after treatment.
Data are mean  SD (n = 40). Different letters indicate significance (P < 0.05, One-way ANOVA). (f) Shoot biomass accumulation, at 17 d after
treatment application. Data are mean  SD (n = 40). Different letters indicate significance (P < 0.05, One-way ANOVA). (g) Wheat stem thickness 15 d
after treatment applications. Data are mean  SD (n = 40). Different letters indicate significance (P < 0.05, One-way ANOVA). (h) Increase in seedling
height over a period of 17 d post inoculation (dpi). (i) Kaplan–Meier plot showing wheat seedling survival rates under the different treatments of YZ1, Fg
(105) and Fg (109) (P < 0.001, Gehan–Breslow–Wilcoxon test). (j) and (k) Relative fungal biomass in stem and roots of wheat plants 17 dpi was measured
by reverse transcription polymerase chain reaction using primers specific for Fg Tri5 (j) and YZ1 VFPJ 08779 (k) and normalized to the wheat actin gene.
Data are mean  SD (n = 10). Different letters at the top of the bars indicate significance (P < 0.05, Student’s t-test).

percentage of FHB-infected spikelets in the YZ1-treated plants was were inoculated with 10 ll of a conidial suspension of Fg. We
5.6  3.4%, while it was 19.15  2.9 in the control (Fig. 3c). found that the percentage of Fg-infected spikes in the YZ1-
To further probe the ability of YZ1 to protect wheat against applied field was 9.7  1.2% while that of the control plants was
FHB under field conditions, an experiment was performed in 42.7  2.3% (Fig. 3d–f). Meanwhile, the number of susceptible
Shijiazhuang in 2023. At the early anthesis stage, wheat spikes spikes were reduced by 89.0% with the field application of YZ1.

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Fig. 3 YZ1 inoculation in roots enhances resistance against Fusarium head blight (FHB). (a) Wheat spikes of YZ1-inoculated plants affected by FHB under
glasshouse conditions. (b) Wheat spikes of non-inoculated plants affected by Fg under glasshouse conditions. All photographs were taken at 14 d after
inoculation with Fg spores. (c) Percentage infected spikes on Fg-inoculated spikelets under glasshouse conditions (n = 20). (d) Wheat spikes of YZ1-
inoculated plants affected by Fg under field conditions. (e) Wheat spikes of non-inoculated plants affected by Fg under field conditions. All photographs
were taken 21 d after inoculation with Fg spores. (f) Relative percentage of infection taken as a measure of susceptibility on Fg-inoculated spikelets under
field conditions (n = 204 for the control and n = 206 for the YZ1-inoculated plants).

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Fig. 4 Antagonistic activity of YZ1 against Fg. (a) Fg mycelium grown on potato dextrose agar (PDA). (b, c) Antagonistic activity of YZ1 against Fg.
(d) Inhibition efficiency of YZ1 ethyl acetate extracts against the growth of Fg on PDA media. (e) Enlargement of dotted box (i) shows morphology of Fg
mycelia growing on a control plate after 20 h. Bar, 25 lm. (f) Enlargement of dotted box (ii) shows morphological characteristics of Fg mycelia growing on
PDA agar medium supplemented with 150 lg ml1 of ethyl acetate extracts from YZ1 after 20 h. Red arrows show Fg mycelia broken by the activity of
YZ1 extracts. Bar, 10 lm. (g) Biocontrol efficacy of YZ1 against Fg was analyzed on wheat seedlings using the Petri dish assay in a growth chamber.
(h) The lengths of wheat leaves. Data are mean  SD (n = 10). Different letters indicate significance (P < 0.05, Student’s t-test). (i) The lengths of lesions
on wheat coleoptiles at 7 d post inoculation were measured. Data are mean  SD (n = 9). Different letters indicate significance (P < 0.05, Student’s
t-test). (j) Correlation between the wheat lesion length and leaf length (P = 0.05). Potato dextrose agar media were used for all plate assays as Fg grew
well in this media. These experiments were repeated a minimum of three times with similar results observed. Different letters at the top of the bars indicate
significance (P < 0.05, Student’s t-test).

YZ1 crude extracts resulted in the inhibition of Fg growth on

YZ1 showed slight antagonistic activity against Fg
PDA media (Fig. 4d). Additionally, our observations suggest that
Fig. 4(a–c) illustrate that YZ1 showed slight antagonistic activity YZ1 extracts caused some degree of breakage in Fg hyphae
against Fg. Moreover, a 20-h treatment with 150 lg ml1 of (Fig. 4e,f).

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Fig. 5 Endophytic colonization of YZ1 in wheat roots. (a) It appears to show a runner hypha on the surface of YZ1-treated roots at 21 h post inoculation
(hpi). Bar, 10 lm. (b) Notching at the point of contact between a fungal hypha and a root hair cell at 8 hpi (white arrow). Bar, 5 lm. (c) Scanning electron
microscopy illustration of early YZ1 colonization and penetration into host plant roots. The image shows YZ1 hyphae (green arrows) penetrating into
intercellular spaces of wheat root epidermal cells (red arrow) and root hairs (white arrow). Bar, 10 lm. (d) Undifferentiated hyphae seen penetrating
between root epidermal cells at 3 d post inoculation (dpi). Bar, 10 lm. (e) Hyphal branches forming on intercellular hyphae (white arrows) at 8 dpi. Bar,
20 lm. (f) Intracellular (yellow arrows) and intercellular hyphae (light blue arrows) colonizing root cortical cells at 7 dpi. Bar, 10 lm. (g) Fluorescence
micrograph of a root cross section taken at 7 dpi showing YZ1 hyphae colonizing the epidermis (e), root cortex (c), and the central cylinder (cc). Bar,
50 lm. (h) YZ1 hyphae seen inside the root central cylinder, xylem. Tracheids are clearly visible in the same focal plane (white arrows) at 16 dpi. Bar, 5 lm.
(i) Outer fungal colonization on a primary root by extraradical hyphae at 8 dpi. Bar, 100 lm. (j) YZ1-GFP hyphae growing inside and emerging from a root
hair at 12 dpi. Bar, 5 lm. (k) Interaction of fungal hyphae with root hairs at 72 hpi. Bar, 25 lm.

Treatment with YZ1 significantly suppressed the disease pro- treated with Fg alone (24.5%). Together, these results show
gression of Fg in germinating wheat seedlings (Fig. 4g). The leaf that YZ1 has slight antagonistic effects on Fg.
lengths of wheat seedlings inoculated with YZ1 and Fg were sig-
nificantly longer than those inoculated with Fg (Fig. 4h). In addi-
YZ1 colonizes wheat endophytically
tion, the lesion lengths on wheat coleoptiles inoculated with YZ1
and Fg were slightly shorter than the coleoptiles of wheat seed- We first generated transgenic YZ1 strains expressing GFP cytoplas-
lings inoculated with Fg alone (Fig. 4i). A correlation between mically (YZ1-GFP) by means of fungal protoplasm transformation.
the wheat coleoptile lesions and the wheat leaf lengths was We observed YZ1 mycelia stained with wheat germ agglutinin
detected (Fig. 4j). A Pearson’s correlation test showed that the (WGA) on the surface of root epidermal cells (Fig. 5a), and on the
severity observed in wheat seedlings treated with YZ1 and Fg surface of root hair cells (Fig. 5b). Using both WGA staining and
(3.6%) was significantly lower (R2 = 0.619, P < 0.05) than that SEM, we found that YZ1 did not form any specialized structures

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when colonizing wheat roots, but the hyphae entered roots by TaPR10) (Fig. S6c,d). Fg treatment alone significantly upregu-
penetrating between root epidermal cells (Fig. 5c,d). WGA staining lated the SA marker gene TaNPR1 compared with the YZ1 + Fg
revealed that YZ1 hyphae spread and entered neighboring cells by treatment (Fig. S6c). By contrast, ET and ABA signaling marker
forming hyphal branches (Fig. 5e). Further, we found that YZ1 genes were not significantly activated in either the YZ1 + Fg or
was able to grow both intra- and intercellularly (Fig. 5f). No Fg treatment at 48 hpi (Fig. S6e–h).
hyphae were observed inside the roots of the non-inoculated plants An additional glasshouse experiment was done to test whether
(Fig S2a–c), but YZ1 was observed in inoculated roots (Fig. S2d– the induction of JA genes persists during the growth period of
f). YZ1 hyphae appeared to colonize and cross through different wheat plants. In the YZ1-inoculated treatment, the expressions of
root cells as free filamentous hyphae (Figs S3f, S4a–c). Some TaAOS1, LIPASE, TaPAL, TaWRKY78, and TaNPR1 were sig-
hyphae were able to cross the suberized cell walls contained in the nificantly higher than in the control group (Fig. 6a–d). SA signal-
periderm and colonize the central cylinder, which contains the vas- ing pathogenesis-related (PR) pathway genes (TaPR2, TaPR1.1,
cular vessels of the plant (Fig. 5g). We therefore checked and con- PR4a, and TaPR10) were not activated by YZ1 inoculation
firmed the presence of YZ1 hyphae inside the root xylem vessels alone, while in the presence of Fg, these genes were significantly
(Figs 5h, S3d). Fluorescent hyphae could be visualized colonizing upregulated (Fig. 6e–i). Furthermore, YZ1 inoculation did not
the surface of the root, inside root hairs, and interacting with root induce other defense genes that were significantly induced by the
hairs (Fig. 5i–k). Fg treatment, namely TaWCI3 (wheat chemically induced1) and
We were able to visualize the presence of many conidia not TaCHI1 (chitinase1) (Fig. 6j,k). Interestingly, we found that
only on the surface of the roots but also inside the cortex cells when plants are inoculated with YZ1, ABA synthesis pathway
and the intercellular spaces (Fig. S3e). Many conidia germinated genes were significantly upregulated, including NCED (9-cis-
inside the cortical cells and inside the root central cylinder cells epoxycarotenoid dioxygenase), TaPLY1 (pyrabactin resistance 1
into germ tubes forming an established point of colonization proteins), and VDE (violaxanthin de-expoxidase) but only in the
(Fig. 3f,g). Inside the metaxylem vessels, many conidia were presence of Fg (Fig. 6l–n). The ET response gene TaPIE was
observed unevenly distributed on the inner walls of these vessels induced by Fg treatment but not by YZ1 inoculation (Fig. 6o).
(Fig. S3h). YZ1 hyphae were observed colonizing both the inter- Meanwhile, we tested the expression of a wheat leaf senescence
cellular and intercellular spaces (Fig. S3i,j). YZ1 hyphae were not marker gene, SAG3 (Chen et al., 2020). We found that this gene
seen on the surface of control plants, but YZ1 profusely colonized was significantly downregulated in YZ1-treated plants, whether
wheat roots (Fig. S3k,l). YZ1 only, or YZ1 in the presence of Fg (Fig. 6p). Further, SPAD
We observed the upward colonization of YZ1 to wheat shoot. measurements showed significantly higher values in plants treated
Green fluorescence was observed in the stem segments of wheat with YZ1 (Fig. 6q). To confirm the significant upregulation of
seedlings (Fig. S4a,b). Additionally, we were able to successfully JA and ABA biosynthesis genes observed in the above results, we
isolate YZ1 cultures from the stem margins, which were con- directly measured the levels of JA and ABA in systemic leaves and
firmed by colony morphology from 30% of the total wheat plants found that they were consistent with the gene expression results.
treated with YZ1 (Fig. S4c), from which the colonies were identi- As SA levels were not elevated, we did not test the levels of SA in
fied by PCR amplification (Fig. S4d). Further, fluorescence leaves. Our analysis revealed that JA levels were significantly
microscopy analysis at 30 dpi confirmed the presence of YZ1 in higher in Fg-treated plants than in YZ1 + Fg-treated plants
healthy leaves, predominantly restricted in leaf veins (Fig. S4e). (Fig. 6r). Similar to the gene expression results, YZ1 + Fg treat-
When senescence of the leaves began at 48 dpi, the entire wheat ments had significantly increased levels of ABA than the non-
leaf was substantially colonized by YZ1 mycelium (Fig. S4f). inoculated control, YZ1, or the Fg treatments (Fig. 6s).
Using quantitative reverse transcription polymerase chain reac-
tion, we detected YZ1 in 8% of healthy leaves at 25 dpi, which
YZ1 modulates wheat root gene expression during early
increased to 35% at 48 dpi in senescent leaves (Fig. S4g).
colonization
We investigated whether YZ1 could systemically colonize the
stems and leaves of wheat under field conditions. The results In total, 12 wheat root samples, comprising six YZ1 samples and
showed that YZ1 could be detected significantly and with high six Fg samples, were collected at 48 hpi and subjected to dual
frequency (Fig. S5). The detection rates of YZ1 in stems and flag RNA-Seq, resulting in 210.08 GB, 116.86 GB, and 110.6 GB
leaves were 19/21 and 16/21, respectively. of clean data, respectively (Table S2). This allowed a comprehen-
sive annotation of transcriptomic changes in the tripartite interac-
tions. The annotation of wheat transcriptomes in this study
Activation of wheat-induced resistance by YZ1 against Fg
identified 209 759 transcripts from a total of 117 759 genes, and
To investigate whether YZ1 activates wheat systemic resistance, 52 125 genes were shared by all the treatments (Fig. 7a). To deci-
we set up a split-root experiment in wheat. YZ1 treatment signifi- pher the expression patterns in bipartite and tripartite setups
cantly upregulated the expression of JA marker genes TaAOS1 between wheat, YZ1, and Fg, we grouped our samples through
and TaOPR by 1.2- and 1.3-fold, respectively, at 48 hpi com- principal component analysis (Fig. 7b). The wheat gene expres-
pared with Fg treatment alone (Fig. S6a,b). Compared with the sion profile induced by all treatments was separated from the
Fg treatment, YZ1 did not significantly upregulate the expression control, while the pattern of YZ1 + Fg is closer to that of Fg
of salicylic acid (SA) signaling marker genes (TaNPR1 and alone than to that of YZ1 alone. Meanwhile, YZ1 led to lower

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Fig. 6 Effects of YZ1 and Fusarium graminearum (Fg) inoculation on the expression of genes associated with different plant hormones 15 d after
inoculation. TaAOS1, TaPAL, LIPASE, and TaWRKY78 are genes associated with jasmonic acid (JA) synthesis in plants (Liu et al., 2016, 2021). TaNPR1,
PR2, PR10, PR1.1, and PR4a are genes associated with salicylic acid (SA) synthesis in plants (Robert-Seilaniantz et al., 2011; Liu et al., 2021). WCI3 and
TaCHI1 are genes involved in general defense response in plants. NCED, VDE, and TaPLY1 are genes associated with abscisic acid (ABA) synthesis in plants
(Chen et al., 2020). TaPIE1 is a gene associated with ethylene synthesis in plants (Zhu et al., 2014; Veselova et al., 2021). SAG3 is a senescence marker
gene in wheat plants (Chen et al., 2020). (a–i) Effects of YZ1 and Fg on the expression of genes associated with JA and SA. (j, k) Effect of YZ1 and Fg on
the expression of genes associated with general defense in wheat. (l–n) Effect of YZ1 and Fg on the expression of genes associated with ABA. (o) Effect of
YZ1 and Fg on the expression of TaPIE, a gene associated with ethylene biosynthesis in plants. (p) Effect of YZ1 and Fg inoculation on the transcription of
SAG3 a wheat leaf senescence marker gene (n = 12). (q) Chlorophyll contents of wheat seedling leaves taken as a measure of SPAD value after 15 d of
treatments (n = 18). (r) Jasmonic acid content in leaves of Fg and YZ1 + Fg treatments at 15 d after treatments (n = 8). (s) Abscisic acid contents in leaves
of Fg and YZ1 + Fg treatments at 15 d after application of treatments (n = 8). Error bars represent the SE of means. Different letters at the top of the bars
indicate significance (P < 0.05, One-way ANOVA).

DEGs (3538) than by Fg (4258), while the treatment YZ1 + Fg encoding components of photosynthesis, mitogen-activated pro-
had the most DEGs (4602) (Fig. 7c). tein kinase (MAPK), suberin/cutin biosynthesis, and hormone
The Gene Ontology (GO) enrichment analysis revealed that signaling pathways were significantly upregulated in YZ1-
genes related to oxidative stress, plant organ senescence, cell wall inoculated plants (Fig. 7f). Moreover, gene-encoding proteins
organization, and aging were notably downregulated in wheat involved in plastocyanin (petE ) photosystem I (PSI), photosys-
roots treated with YZ1 (Fig. 7d). Additionally, DEGs for chitin tem II (PSII), ferredoxin (petF ), and several elements involved in
activity were common in both the YZ1 and Fg treatments light harvesting were significantly upregulated by YZ1 inocula-
(Fig. 7e). Overall, we found that GO terms associated with car- tion (Table S3). It was found that WRKY22 (LOC123162630)
bohydrate synthesis were significantly enriched in YZ1- and WRKY33 (LOC123136873, LOC123059977, LOC
inoculated plants (Fig. S7). KEGG analysis showed that genes 123182356, LOC100049055, and LOC100049024) were

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Fig. 7 Dual RNA-Seq results of the tripartite interactions among wheat, YZ1, and Fusarium graminearum (Fg). Roots were harvested for sequencing at 48
h post inoculation. (a) A Venn diagram showing unique DEGs in each treatment (FDR < 0.05). (b) Principal component analysis of wheat root gene
expression levels during individual or simultaneous colonization by YZ1 and Fg. (c) The total number of upregulated (left bars) and downregulated (right
bars) DEGs between the different treatments used in this study (> log2FC; FDR with adjusted P-value < 0.05), compared with non-inoculated plants. (d)
Gene Ontology (GO) term enrichment analysis of the downregulated DEGs of wheat roots in response to YZ1. (e) KEGG pathway enrichment of wheat
DEGs in response to YZ1. (f) GO term enrichment analysis of the upregulated DEGs of wheat in response to the treatment of YZ1, Fg or both. (g) Key
wheat DEGs of the YZ1 treatment enriched in pattern-triggered immunity immunity following KEGG enrichment analysis. (h) Key wheat DEGs of the YZ1
treatment enriched in MAPK signaling pathway and plant–pathogen interaction pathways. (i) and (j) Key wheat DEGs of the YZ1 treatment enriched in
jasmonic acid and salicylic acid metabolism, respectively, following KEGG analysis. (k) Expression of general defense response genes, plant–pathogen
related, and plant hormone signaling pathway genes examined by quantitative polymerase chain reaction. Error bars represent the SE of means (n = 3).
Asterisks at the top of the bars indicate significance (P < 0.05, Student’s t-test).

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significantly upregulated in YZ1-inoculated plants (Fig. 7g,h; treated plants was significantly higher than that of the flag leaves
Table S3). JAZ, a gene that negatively regulates JA biosynthesis, of non-treated plants (Fig. 8e).
was significantly downregulated (Fig. 7i), while SA metabolism We found that the cell walls of wheat root colonized with YZ1
genes NPR1 and PR-1 were significantly upregulated in the YZ1 were thickened significantly (Fig. 8f,h). Statistics of the imaging
treatment (Fig. 7i,j). Quantitative polymerase chain reaction ana- results demonstrated an 80% increase in cell wall thickness of
lysis further confirmed the significant upregulation of SA, JA, wheat by YZ1 inoculation. Using Calcofluor White staining,
and general defense response genes in the YZ1 treatment at 48 which labels beta-linked polysaccharide in the plant cell walls
hpi (Fig. 7k). blue including cellulose, we observed substantial removal of
We analyzed GO terms of the uniquely induced DEGs in each cellulose from the wheat root cell walls inoculated with Fg, but
treatment. In total, we found 1730 unique DEGs upregulated in no significant differences were observed between mock and YZ1-
wheat roots in the YZ1 treatment, much fewer than the 3355 inoculated root tissues (Fig. 8i–k).
unique DEGs in the Fg treatment (Fig. S8a). GO analysis
showed that photosynthesis and biological processes in interspe-
Discussion
cies interactions were significantly enriched in wheat roots colo-
nized by YZ1 (Fig. S8b). In the Fg-treated wheat, GO terms Fusarium diseases, particularly FHB, are devastating to wheat
associated with carbon lyase activity, xylan catabolic process, and production world-wide (Parry et al., 1995). The pathogen strain
cell wall organization were significantly enriched (Fig. S8c). Simi- used in this study, F. graminearum, is one of the most virulent
larly, in the YZ1 + Fg treatment, GO terms associated with plant variants causing disease in many cereal crops (Xu et al., 2022).
cell wall organization, cell wall catabolic process, and defense This strain caused rapid wheat death at the seedling stage
response to fungi were significantly enriched (Fig. S8d). Overall, (Fig. 2a,b,i) and severe FHB at the maturity stage (Fig. 3) as
these results suggest that the wheat transcriptome in response to tested in this study. Currently available BCAs against Fusarium
colonization by YZ1 differs from that in response to attack by the diseases are believed to be primarily based on the antagonistic
pathogen Fg. effects (Besset-Manzoni et al., 2019; Cantoro et al., 2021; Feng
et al., 2023; Whitaker et al., 2023). This is primarily due to the
fact that previous research has predominantly concentrated on
Genes encoding plant cell wall degrading enzymes are
antagonistic mechanisms, with minimal exploration of induced
upregulated in YZ1
resistance. In the case of specific biocontrol products such as Ser-
GO term enrichment analysis showed that the transcriptomes of enade ASO (EFSA et al., 2021), the initial belief was that protec-
YZ1 and Fg were significantly different during in-planta coloni- tion was conferred through metabolites upon application.
zation. Major GO terms enriched in YZ1 included integral and Nevertheless, it is now recognized that the product also elicits
intrinsic components of the membrane (Fig. S9a), whereas in Fg, induced resistance. While biocontrol shows promise for sustain-
glycoprotein and terpenoid metabolic processes were induced able agriculture, biocontrol strains based on antagonism are sus-
(Fig. S9b). GO terms associated with plant cell wall degrading ceptible to environmental changes in field conditions.
enzymes (PCWDEs) were significantly enriched in YZ1 during Consequently, endophytic strains have emerged as a more robust
endophytic colonization inside wheat roots, especially hydrolase biocontrol approach, as the internal plant environment offers
activity (Fig. S10a). We found that a number of PCWDEs, parti- endophytes a secure and stable habitat with essential resources for
cularly glucan-13-beta-glucosidase (VFPFJ_09277), were signifi- survival (Larran et al., 2016). Here, we report a growth-
cantly enriched in YZ1 during in-planta colonization of wheat promoting fungus YZ1, which is a potent endophyte and capable
(Fig. S10b). of safeguarding wheat against Fusarium infection primarily by
inducing systemic resistance in wheat.
The potent nature of endophytism may be a significant advan-
YZ1 colonization increased wheat photosynthesis and cell
tage of YZ1 in influencing wheat growth, providing systemic pro-
wall thickness
tection, thereby suppressing Fusarium. This colonization process
RNA-Seq results implied that YZ1 inoculation enhanced photo- can be rapid at all wheat growth stages of dormant seed, seedling
synthesis (Fig. 8a,b), saccharide biosynthesis, and cell wall and spiking, as confirmed by PCR detection (Figs S4d, S5a,b),
remolding in wheat (Fig. S7). We therefore further examined quantitative polymerase chain reaction (Figs 2j,k, S4g) and var-
photosynthesis-related parameters and cell wall thickness in YZ1- ious imaging results (Figs 5a–k, S2, S3). Upon colonizing the
inoculated wheat. root surface, YZ1 penetrates the roots through root epidermal
In the flag leaves, YZ1-treated plants had a significantly higher cells and colonizes cortical cells intra- and intercellularly and
total Chl content (62.0  2.2 lg mg1) than the Chl contents overcomes the suberin layer to colonize the central cylinder. The
of non-treated control plants (58.3  3.4 lg mg1) colonization process by YZ1 is notably rapid. At the seedling
(P < 0.0002) (Fig. 8c). A similar pattern was seen for the total stage, the YZ1 hyphae can reach the wheat stem within c. 8 d
carotenoid content at the anthesis stage. The total carotenoid and can colonize leaves massively in around a month. Our obser-
content of YZ1-treated plants was significantly higher vations suggest that this colonization can persist until the matur-
(P < 0.0004) than that of the untreated plants (Fig. 8d). Further, ity stage in both laboratory and field conditions within the wheat
we found that the photosynthetic rate of the flag leaves of YZ1- plant (Fig. S5). Persistence of the strain YZ1 in the field was also

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observed in our previous studies (Yang et al., 2022). This level of rapeseed leaves after 30 d (Zhang et al., 2020). From a disease
persistence is comparable to imaging results of Colletotrichum control perspective, it might be advantageous for an organism to
tofieldiae, which was able to heavily colonize Arabidopsis leaves in have a prolonged survival in the environment; however, this is
28 d (Hiruma et al., 2016), and Sclerotinia sclerotiorum, which generally not considered beneficial. The potential negative
was detected in wheat stems at 45 d (Tian et al., 2020) and impacts of BCAs on the environment and biodiversity must be

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Fig. 8 YZ1 colonization induced enhanced photosynthesis and cell wall thickening in wheat. (a, b) Gene clusters and Gene Ontology (GO) terms induced
by YZ1 inoculation based on the RNA-Seq results. (c) Total Chl content in flag leaves at anthesis stage (n = 10; Student’s t-test, P < 0.001). (d) Total
carotenoid content of flag leaves at anthesis stage (n = 10; Student’s t-test, P < 0.0002). (e) Photosynthetic rates of flag leaves in non-inoculated plants
and YZ1-inoculated plants at anthesis stage (n = 12). (f) Plant cell wall thickness at 96 h post inoculation (hpi). Root cross section of mock-inoculated
wheat roots (g) and YZ1-inoculated roots (h) showing cell wall thickness 96 hpi. Bars, 5 lm. Wheat roots of mock-inoculated (i), Fg-inoculated (j), and
YZ1-inoculated (k) stained with 0.01% Calcofluor white, which labels beta-linked polysaccharides in plant cell walls blue (including cellulose). Identical
microscope settings were used in this experiment. Bars, 25 lm.

thoroughly assessed. Based on our previous research (Yang microbiomes can adequately persist in a plant-microbiome breed-
et al., 2022), YZ1 has been observed to persist in soil without sig- ing experiment, providing beneficial responses to selection (Han-
nificantly altering the community structure and diversity. nula et al., 2021). However, it is now well-established that plant
Furthermore, YZ1 is recognized as a native soil fungus. Conse- microbiomes are influenced not only by genotype but also by
quently, its ecological safety might not currently be a concern. environmental microbes (Latz et al., 2021). Therefore, more in-
The double-pot inoculation experiment demonstrated that depth studies are required to fully adopt microbiome breeding
YZ1 could successfully colonize wheat shoot through exclusive (Wei & Jousset, 2017).
root application (Fig. S4a–g). Different from many antagonistic It has been recognized that many beneficial microbial species
biocontrol agents, YZ1 as a stringent endophyte offers a unique can both promote plant growth and induce systemic resistance,
feature for applying YZ1 as a typical fertilizer for root uptake. although connections between these two effects remain unclear
This distinctive trait allows for the incorporation of live YZ1 with (Lacava et al., 2022; Pellegrini et al., 2023). The results presented
carriers such as biochar, simplifying the application process (Yang in this study indicated that the robust systemic resistance against
et al., 2022). The utilization of immobilization technology holds Fg may be a result of the mitigation of the growth-defense trade-
promise for the industrial-scale production of biocontrol agents off in wheat by YZ1. Plants have evolved a mechanism for
(Wu et al., 2021). We speculate that the rapid, systemic, and growth-defense trade-offs, and one way to mitigate the trade-off
enduring endophytic colonization exhibited by YZ1 is a key fac- is to unlock the growth and restrict the expression of resistance
tor contributing to its effectiveness in promoting growth and sup- (Wang et al., 2021; He et al., 2022). In this study, Fg induced a
pressing Fusarium under field conditions. bundle of defense genes, catabolic processes (Fig. 7f), and cell
We observed a slight antagonistic effect of YZ1 on Fg. This wall damages. We observed a distinct impact of YZ1 colonization
antagonistic effect was observed on the plate (Fig. 4a–c), in the on wheat. YZ1 behaved like a typical beneficial endophyte, exhi-
rhizosphere (data not shown) and inside the wheat root (Fig. 2j, biting reduced ROS production (Fig. 7c) and unlocked wheat
k), possibly due to its antimicrobial activities (Fig. 4d–f). The sup- growth (Figs 1, 2c–h, 7f). It is important to highlight that field
pression of Fg in wheat seems to be primarily driven by the strong tests conducted in Baoding and Yancheng, China, demonstrated
induced resistance by YZ1 colonization rather than its antimicro- a significant yield increase of 3.7–11% after a single application
bial effect. Compared with the reported strains such as Tricho- of YZ1. These results were partially presented by Yang
derma guizhouense NJAU4742 (Tao et al., 2023), YZ1 does not et al. (2022) and further supported by additional data not shown
exhibit sufficient potency to serve as a biocontrol agent solely here. Overall, YZ1 could substantially promote growth and
based on its antagonistic effect. Upon colonization, YZ1 triggered enhance wheat yields. Interestingly, the increased photosynthesis
a significant increase in the plant’s immune response (Fig. 6a–p), rate may be a key factor contributing to the growth promotion
and this induction appeared to be long-lasting in terms of both by YZ1, as evidenced by higher Chl content (Fig. 8c–e) and the
gene expression and pathogen suppression. This induction had a significant upregulation of genes related to the photosynthesis
systemic effect, considering the solid evidence that exclusive root pathways (Figs 7f, 8a,b). The findings suggest that the enhanced
inoculation induced a sharp increase in expression of defense photosynthesis may have facilitated energy production and sac-
genes in shoot (Fig. S6a–h) and strong pathogen suppression in charide synthesis, supporting cell wall thickening in wheat as a
spikelets in both the pot culture and the field trial (Fig. 3). Our defense response against Fg (Fig. 8f–h). It has been reported that
tests further showed that the resistance effect was not specific to fungal invasion can trigger the plant’s recognition of damage-
Fg alone, but also against powdery mildew in wheat (Fig. S11). associated molecular patterns (DAMPs) (Ning et al., 2017), while
These characteristics align with the strict definition of induced YZ1 propagation within wheat was found to breach the cell wall
resistance as being distantly interactive, non-specific, and long- (Fig. 5f) with an upregulation of YZ1 PCWDEs (Fig. S10a).
lasting (Heil & Bostock, 2002). Induced resistance has been dis- These DAMPs may serve as alarm signals, activating the plant’s
covered long ago, and a number of plant growth-promoting fungi immune response, particularly cell wall thickening, a defense
have been identified having such ability (van Loon et al., 2006; mechanism observed in response to various fungal species (Anil
Lugtenberg & Kamilova, 2009). Currently, pyramiding elite et al., 2013).
alleles for combating pathogens is becoming increasingly difficult
(Li et al., 2020), while effective resistance genes for Fusarium dis-
Conclusions
eases are relatively scarce (Zhu et al., 2019). Instead, microbiome
breeding may offer a new approach for sustainable disease resis- The ultimate measure of success for a biocontrol agent lies in its
tance. Research has demonstrated that chosen host-associated ability to perform effectively under real-world production

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16 Research Phytologist

conditions (Fravel, 2005). Through multiple rounds of culture manuscript. RNK, XZ and XL finished data analysis. All authors
experiments, we consistently demonstrated YZ1’s strong capabil- revised and approved the paper. RNK and XZ contributed
ities in promoting growth and suppressing Fg. Moreover, the suc- equally to this work.
cessful industrialization of biocontrol agents necessitates the
incorporation of superior traits and collaborative efforts across
disciplines. These strains should possess properties of rapid ORCID
growth, ecological safety, and the ability to persist in field condi- Xiaofang Li https://orcid.org/0000-0003-1554-4484
tions (Lewis & Papavizas, 1991). The ecological safety of YZ1 Xin Zheng https://orcid.org/0000-0002-3402-8680
has yet to be systematically assessed, while the efficacy and adapt-
ability of YZ1 in soil were tested in this study. The presence of
YZ1 and the protection in wheat plants were found to endure for Data availability
the duration of the testing period. Our previous results showed All primers can be found in Tables S1–S3. Raw transcriptomic
that YZ1 can sustain in wheat rhizo-soil for at least 2 months in data can be accessed under the GSA accession no. CRA012800
the field (Yang et al., 2022). This suggests that YZ1 may possess (BioProject: PRJCA020149) on the website of National Geno-
a significant competitive advantage over indigenous microbial mics Data Centre (https://ngdc.cncb.ac.cn/).
communities, enabling its successful establishment and persis-
tence in both the rhizosphere and endosphere.
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