Pesticide Biochemistry and Physiology 190 (2023) 105316

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Pesticide Biochemistry and Physiology

journal homepage: www.elsevier.com/locate/pest

Application of Ceriporia lacerata HG2011 as biocontrol agent against

multiple phytopathogenic fungi and oomycetes
Jie Yin , Ruxia Bai , Ling Yuan , Jian-guo Huang *
College of Resources and Environment, Southwest University, Chongqing 400716, China

A R T I C L E I N F O A B S T R A C T

Keywords: Overuse of fungicides to control crop diseases results in ecological damage, environmental pollution, and human
White-rot fungus health risks. Biocontrol is an increasingly popular alternative in plant disease management due to sustainability
Antibiosis and environmental friendliness. Herein, antagonistic tests and greenhouse experiments were conducted to
Mycoparasitism
investigate the antagonism of a self-isolated white-rot fungus Ceriporia lacerata HG2011 against phytopathogens
Cell wall degradation
Volatile compounds
in vitro, the underlying mechanism exerted by this fungus, and disease control efficiency in the greenhouse. The
results demonstrated that both soluble and volatile substances produced by this fungus suppressed the growth of
all test phytopathogen fungi and oomycetes in vitro, with the inhibitory rates of 10.4–60.6% for soluble me­
tabolites and 30.3–52.9% for volatiles. C. lacerata HG2011 could grow in and gradually spread on living
phytopathogenic colonies, concurrently deformed and lysed pathogenic hyphae in dual culture, which were
associated with the release of hydrolase (cellulose, chitinase, β-glucanase, and protease) from this biocontrol
fungus for the use of the pathogens as nutrient sources. The chitinolytic and cellulolytic production by C. lacerata
HG2011 presents the specific response to the cell wall of pathogenic fungi and oomycetes, and β-glucanase was
triggered by carbon competition. Consequently, C. lacerata HG2011 successfully controlled eggplant stem blight
and cucumber vine blight (control efficacy 67.9–70.9%) in the greenhouse experiments. C. lacerata HG2011
showed multiple antagonistic mechanisms against the phytopathogenic fungi and oomycetes concurrently. Our
results provided information about a new potential use of this fungus as a biocontrol agent to control plant
diseases in modern agriculture beyond medical purposes, wastewater treatment, and biofuel production.

1. Introduction conscious, and sustainable in plant disease management (Tariq et al.,

2020).
Plant diseases are considered one of the most important limiting The suppression of plant diseases by Pseudomonas spp., Bacillus spp.,
factors in crop cultivation apart from nutrients. Intensive cropping in Lysobacter spp., and Streptomyces spp. has long been studied (Stein,
modern agriculture promoted the occurrence of plant diseases, leading 2005; Breidenstein et al., 2011; de Lima et al., 2012; Panthee et al.,
to 30–70% yield loss worldwide (Mishra et al., 2016; Savary et al., 2016). The underlying mechanisms employed by biocontrol microor­
2019). The application of synthetic pesticides, the breeding of disease- ganisms involved in crop disease suppression include the production of
resistant varieties, and better field management are traditional strate­ antimicrobial substances against phytopathogens, competition with
gies to control crop diseases in crop cultivation (Barzman et al., 2015; phytopathogens for nutrients and living space, and induction of systemic
Raymaekers et al., 2020). Although synthetic pesticides can effectively resistance in plants against different phytopathogens, etc. (Beneduzi
control plant diseases, they produce many harmful impacts on ecosys­ et al., 2012; Ghorbanpour et al., 2018). However, most biocontrol mi­
tems, non-target organisms, and even humans (Mihajlović et al., 2017). croorganisms were often apt to lose their biocontrol potential under
The long-term and large application of synthetic pesticides makes open field conditions (Gerbore et al., 2014). Based on the known liter­
pathogens drug-resistant (Yaqub et al., 2018; Ernst et al., 2018) and thus ature, quantities of known antimicrobial substances produced by fungi
declines pesticide efficiency (Bhandari et al., 2019). Compared with were about 4900, far more than 2900 produced by bacteria (Bérdy,
chemical pesticides, biological control is human-safe, ecology- 2005). In addition, biocontrol fungi present strong viability and a

Abbreviations: VCs, volatile compounds; CLI, C. lacerataHG2011 solid inoculant; CEFB, the crude extract of fungal broth produced byC. lacerataHG2011.
* Corresponding author.
E-mail address: [email protected] (J.-g. Huang).

https://doi.org/10.1016/j.pestbp.2022.105316
Received 24 September 2022; Received in revised form 3 December 2022; Accepted 7 December 2022
Available online 9 December 2022
0048-3575/© 2022 Elsevier Inc. All rights reserved.
J. Yin et al. Pesticide Biochemistry and Physiology 190 (2023) 105316

variety of mechanisms to antagonize plant pathogens compared with 2. Material and methods
biocontrol bacteria (Tariq et al., 2020). The suppressive efficacy of
biocontrol fungi such as Pythium oligandrum (Gerbore et al., 2014), 2.1. C. lacerata HG2011 and phytopathogen
Trichoderma spp. (Verma et al., 2007), and Chaetomium globosum (Hung
et al., 2015) have been intensively studied against many diseases incited C. lacerata HG2011 (GenBank accession No. MT675050) was
by foliar and soil-borne phytopathogens. deposited at the China Microbiological Culture Collection Center
The cell walls of many plant pathogens contain β-glucan, chitin, and (Wuhan) and also maintained at the College of Resources and Envi­
or cellulose as a structural backbone and other minor components such ronment, Southwest University. The suspension of C. lacerata HG2011 (i.
as proteins, phospholipids, and lipids (Geoghegan et al., 2017). There­ e. SCL, 1 × 105 CFU mL− 1) and C. lacerata HG2011 solid inoculant (CLI,
fore, lytic enzymes such as chitinase, cellulose, β-glucanase, protease, approximately 2.1 × 108 CFU g− 1) were used in the greenhouse exper­
phosphatase, and lipase may decompose those components in pathogen iments. To prepare SCL, 5 plugs of mycelia 6 mm in diameter were cut
cell walls and thus are involved in the mycoparasitic process of many from the young edge of fungal colonies on PDA, inoculated in 500 mL
biocontrol microbes (Hasan et al., 2014). A large proportion of sterile PDB, and incubated for 5 days at 28 ◦ C with agitation at 120 rpm
biocontrol bacteria can biosynthesize and then release only one or a few in the dark. The 5-day-old culture fluid was then transferred to a
antimicrobial substances and therefore present a limited biocontrol ef­ fermentation tank (BailunBio, Shanghai Bailun Biotechnology Co., Ltd.)
fect. Besides, very few genera of biocontrol fungi and bacteria are containing 20 L modified glucose peptone yeast (GPY) medium and
currently used in plant disease management. For instance, only 14 incubated for 7 days with aeration at 0.01 vvm at the same temperature
biocontrol microbes were registered in European Union (Gerbore et al., and agitation as abovementioned. Thereafter, large mycelia were
2014) and 24 microorganisms have been included in Annex I of directive removed via a cheesecloth. To prepare solid C. lacerata inoculant (CLI),
91 / 414, which lists the active substances authorized for use in plant the abovementioned fermentation liquid was inoculated in the culture
protection (Alabouvette et al., 2009). Hence, it is necessary to seek substance (1:4, v:w) consisting of water, rice husk, and corn flour at the
efficient biocontrol microorganisms that can be used in practice. proportion of 50: 49: 1 (weight: weight: weight) and incubated at room
Ceriporia lacerata, a white-rot fungus, grows on live and dead trees in temperature (RT, 22–28 ◦ C) in the dark until the hyphae occupied all
some boreal forests and uses lignin, cellulose and protein as carbon and substrate. The CLI can store at RT for at least 3 months without vitality
nutrient sources via the production of extracellular enzymes, such as decrease.
manganese peroxidase, laccase, lignin peroxidase, cellulase, protease The phytopathogens used in the present experiment included Fusa­
and phosphatase (Tang et al., 2010; Jang et al., 2012; Sirén et al., 2015). rium graminearum (infects wheat), Colletotrichum gloeosporioides (infects
This fungus has been traditionally used for medicinal purposes, waste­ citrus), Rhizoctonia solani (infects barly and wheat), Botrytis cinerea
water treatment, and industrial production of sugar and biofuel (Kumar (infects tomato), Didymella bryoniae (infects watermelon and cucumber),
et al., 2009; Zhang et al., 2015; Yu et al., 2018). Recently, some re­ Alternaria alternata (infects tobacco), Phytophthora nicotianae (also in­
searchers found that C. lacerata can also live in plant tissues and rhi­ fects tobacco), and Phytophthora capsici (infects pepper and eggplant).
zospheres (Paul et al., 2006; Wang et al., 2013; Niu et al., 2017). Some All of them were generously provided by the Institute of Plant Ecological
antimicrobial properties substances have been isolated and identified Pathology, Southwest University.
from C. lacerata culture solution, including 2′ ,4′ -dihydroxy-6′ -methoxy-
3′ ,5′ -dimethylchalcone, huperzine A, mannosylerythritol lipid, lano­ 2.2. Antimicrobial activity of soluble compounds produced by C. lacerata
stane triterpenoids, tremulane sesquiterpenes, furan derivatives and so HG2011
forth (Özçelik et al., 2011; Zhao et al., 2013; Niu et al., 2017; Yu et al.,
2018). Sreeja et al. (2016) isolated a new strain of C. lacerata from black To obtain the crude extract of fungal broth produced by C. lacerata
pepper plants with the ability to antagonize P. capsici in vitro. Consid­ HG2011 (CEFB), 1 L of 7-day-old C. lacerata HG2011 fermentation
ering the facts above mentioned, it is reasonable to speculate that some liquid was sequentially passed through sterile gauze and a 0.22 μm-
C. lacerata strains might inhibit plant pathogens (not only P. capsici) via aperture filter to remove fungal hyphae, spores, and other solids. The
the release of antibiotics and lytic enzymes that degrade pathogen cell fungus-free filtrate was then extracted with ethyl acetate in a rotary
walls and thus effectively reduce plant disease incidence. However, evaporator under reduced pressure at 25 ◦ C to obtain the crude extract.
there is a lack of evidence supporting our hypothesis. The crude extract of different weights was dissolved in 5% methanol and
A novel strain of C. lacerata (HG2011) was initially isolated from the then mixed with PDA (prior to solidification) to arrive at nominal con­
Jinyun National Forest Park in Southwest China and identified by centrations of 0 (fungal broth was supplemented with methanol alone;
morphology and molecular phylogenetic analyses (Yin et al., 2021a). In CK), 0.2, and 0.4%, respectively. After solidification, a mycelial disc 5
our early experiments related to organic nutrient mobilization, this mm in diameter was cut from the edge of young phytopathogen col­
fungus produced extracellular hydrolases (cellulase, protease, phos­ onies, inoculated at the plate center, and incubated for 4 days in the
phatase, lipase, and amylase) and siderophore in culture mediums and dark. Radial mycelial growth was recorded and percent inhibition was
soils, increasing plant nutrient uptake, biomass, and yield for several calculated as: [(DCK-D)/DCK] × 100 (Saravanakumar and Wang, 2020).
crops like eggplant, tomato, broad bean, etc.(Yin et al., 2021a, 2021b; where D and DCK are the diameter (cm) of the pathogen colonies with
Yin et al., 2022). In the present study, we assessed the antimicrobial and without fungus-free broth culture added. The tests were performed
potential of C. lacerata HG2011 against six different pathogenic fungi in five replications.
and two oomycetes in vitro, revealed the possible antagonistic mecha­
nisms, and then extended the study to greenhouse plants by comparing 2.3. Antimicrobial activity of volatile compounds (VCS) produced by
disease control efficacies by this fungal species with synthetic pesticides. C. lacerata HG2011
The main objectives were to (i) confirm our hypothesis that C. lacerata
HG2011 may have an inhibitory effect on plant pathogens, (ii) under­ The antagonistic effect of VCs on pathogens was evaluated using the
stand the underlying mechanisms employed by this fungus involved in sealed plate method with modifications (Fernando et al., 2005). A 5-mm
antagonism against these phytopathogens, and (iii) evaluate the po­ plug of phytopathogen and C. lacerata HG2011 cut from the actively
tential use of this fungus as biological control agents in crop disease growing cultures were placed on the center of a PDA plate in two bases
management. of Petri dishes, respectively. Then two dishes were taped together with
parafilm. Control treatment (CK) was set up in the same way with blank
plates instead of C. lacerata HG2011 dishes. There are 5 repetitions in
each treatment and all plates were incubated at 25 ◦ C in darkness. The

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J. Yin et al. Pesticide Biochemistry and Physiology 190 (2023) 105316

hyphal growth of the phytopathogens treated with and without infecting eggplant (cv. Sanyueqie) and D. bryoniae on PDA for infecting
C. lacerata HG2011 were observed by digital microscope (Mshot MC56, cucumber (cv. Jinyou1) at 25 ◦ C for 7 days in the dark. Spores were then
Guangzhou Mingmei Photoelectric Technology Co., Ltd., Guangzhou, collected from the plates in sterile distilled water and diluted to 1 × 105
China). The radial growth of test pathogens was compared at 5-days spores mL− 1 (checking by a hemocytometer).
incubation and percent inhibition was calculated as: [(DCK-D)/DCK] × Typical Udorthent (loam texture, pH 6.51) was placed in pots
100. (diameter × height = 15 cm × 17 cm, volume = 3.0 dm3, 2.0 kg soil per
where D and DCK are the diameter (cm) of the pathogen colonies with pot) was fertilized with adequate N, P and K (0.2 g (NH2)2CO and 0.2 g
and without C. lacerata HG2011, respectively. KH2PO4 kg− 1 soil). Each disease control experiment was conducted
twice in the greenhouse using a randomized complete block design with
2.4. Mycoparasitism and the hyphal interactions 3 replications for each treatment (30 pots per replication). Experimental
treatments included: (i) seedling alone (CK), (ii) pathogen inoculation
The mycoparasitism of C. lacerata HG2011 on plant pathogens was (PI), (iii) pathogen inoculation plus chemical fungicide (PI+CFC), (iv)
evaluated using a dual culture test (Abdel-Rahim and Abo-Elyousr, pathogen inoculation plus C. lacerata HG2011 inoculation (PI+CL). 4-
2018). A 5-mm-diameter disk of pathogen and C. lacerata HG2011 week-old seedlings of eggplant and cucumber were individually trans­
were inoculated at the bisector 3 cm away on a PDA plate with 3 rep­ planted into pots (one seedling per pot). P. capsici mainly infected the
lications (Fig. 3) and incubated at 25 ◦ C in the dark. The growth of joint between the stem and root and D. bryoniae chiefly infected leaves.
phytopathogens and C. lacerata were daily checked and photographed To suppress the joint infection of eggplant, 50 mL of P. capsici spore
after incubating for 3, 5, and 7 days. suspension was evenly mixed with 2 kg soil in each pot in the PI, PI+CL,
The hyphal interactions between C. lacerata HG2011 and pathogens and PI+CFC treatments. One day later, 25 g CLI was placed in the soil
were investigated according to Abdel-Rahim and Abo-Elyousr (2018) immediately around each eggplant seedling at the time of transplanting
with modifications. A glass coverslip coated with 1 mL of sterilized 2% in the PI+CL treatment. PI+CFC was performed in the same way but
agar solution was placed on solidified water agar plates. One 5 mm- using 30 mL of 3000-fold-diluted mandipropamid (REVUS®, Syngenta,
diameter plug of C. lacerata HG2011 and tested phytopathogen were Switzerland) instead of CLI. In the foliar disease control experiment,
individually inoculated on the two opposite sides of the coverslip, and 100 mL of D. bryoniae spore suspension was sprayed on the whole
then the plates were incubated at 25 ◦ C with darkness. After two mi­ seedlings of cucumber in the PI, PI+CL, and PI+CFC treatments after 7
crobes met each other for 24 h, the slides were taken out to observe the days of transplanting. Two days later, the cucumber seedlings were
hyphae in the interaction zone of two microbes by using a digital mi­ sprayed with SCL in the PI+CL treatment, 800-fold-diluted thiophanate-
croscope (Mshot MC56, Guangzhou Mingmei Photoelectric Technology methyl (Nisso® TOPSIN-M, Nippon Soda Co., Ltd. Japan) in PI+CFC,
Co., Ltd., Guangzhou, China). and sterile water in CK until runoff, respectively. Plants were maintained
in the greenhouse under natural light and temperature with watering as
2.5. The use of pathogen cell walls by C. lacerata HG2011 as nutrient needed every 2–3 days. Disease incidence was recorded daily until
sources reached about 35% - 50% in PI. At this time, plant disease classes were
scored based on Foster et al. (2013) for eggplant blight and Gusmini
The cell wall of plant pathogens was prepared based on the method et al. (2005) for cucumber gummy stem blight, respectively. The disease
developed by López-Mondéjar et al. (2011). Briefly, P. capsici, A. alter­ severity index (DSI) and control efficacy (DCE) in all treatments were
nata, and D. bryoniae were individually grown in potato dextrose broth calculated by the method of Rekanovic et al. (2007):
(PDB) in a rotary 150 for 10 days. The mycelia were harvested by (∑ )/ )
filtration with sterile gauzes, lyophilized, and powdered. Three myce­ DSI = 100 × C × NC BC × NT
lium plugs of C. lacerata HG2011 5 mm in diameter were inoculated into
150-mL Erlenmeyer flasks containing 50 mL PDB with decreased glucose DCE = 100 × (DSICK − DSIT )/DSICK
(0.4% glucose, pH 6.5). C. lacerata HG2011 varied little, if any at all, in
biomass when grown in PDB with glucose at 0.4% and 1% (normal where C = Disease class (0, 1, 2, 3, 4…), NC = Number of plants in
concentration). Thereby seven treatments were set up, including a corresponding disease class, BC = The biggest disease class, NT = Total
sugar-decreased PDB (0.4% glucose) without pathogens as a control, number of plants in each treatment, DSICK = disease severity index in CK
PDB with cell wall powder of plant pathogens (P. capsici, A. alternata, and DSIT = disease severity index in treatment.
and D. bryoniae, respectively; weight: volume) at the concentration of
0.4% and with no glucose, and PDB with 0.4% glucose and 0.4% path­ 2.7. Statistical analysis
ogen cell walls. Hereafter, the treatments are referred to as CK, P. capsici
CW, A. alternata CW, D. bryoniae CW, P. capsici CW + glucose, All the experimental data were subjected to two- or one-way analyses
A. alternata CW + glucose, and D. bryoniae CW + glucose, respectively. of variance using SPSS 23.0 software (SPSS Inc., Chicago, IL, USA). Since
All flasks were incubated at 25 ◦ C with shaking at 150 rpm in the dark. the trend of the two experiments was the same, the data obtained in the
There were 4 replicate flasks for each treatment. After incubating for 5 latest greenhouse experiment was used in this paper. Duncan’s multiple
days, the culture solutions in each flask were collected by centrifuge at range test was used to determine significant differences between the
10000 g, 4 ◦ C for 10 min. The supernatants were analyzed for the chi­ treatment at P ≤ 0.05 for all experiment.
tinolytic, β-glucanolytic, cellulolytic, proteolytic and mono­
phospholipidlytic activities of C. lacerata HG2011 on colloidal chitin, 3. Results
laminarin (a storage glucan), sodium carboxymethyl cellulose, casein,
and lecithin, respectively (Halstead, 1964; König et al., 2002; Qualhato 3.1. Antagonistic assay of metabolites from C. lacerata HG2011 in vitro
et al., 2013; Prakash et al., 2014).
As shown in Fig. 1, the crude extracts of fermentation broth (CEFB)
2.6. Biocontrol of plant diseases prepared from C. lacerata HG2011 inhibited the mycelial growth of the
eight tested phytopathogens in a dose-independent manner. The inhi­
To investigate biocontrol efficacy of C. lacerata HG2011 in planta, bition rate was 24.6–60.6% at 0.4% CEFB.
most studied oomycetes P. capsici and fungi D. bryoniae were selected to After 5-day incubation, volatile compounds produced by C. lacerata
infect the corresponding crops in the greenhouse experiments. P. capsici HG2011 (VCs) also demonstrated a significant antagonistic effect on the
was grown on an oat agar medium (3% oat powder and 2% agar) for mycelial growth of all test phytopathogens in both horizontal and

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Fig. 1. Antagonistic activity of the crude

extracts of fungal broth prepared from
C. lacerata HG2011 (CEFB) against plant
pathogens tested in vitro. (A) Changes in the
colonies of phytopathogens on PDA con­
taining CEFB at different concentrations
after 4-day incubation. (B) Inhibition per­
centage of phytopathogenic colonies treated
with different concentrations of CEFB. Bar
represents standard error and the distinct
small letters on the bar indicate significant
differences across treatments at P ≤ 0.05
(Two-way ANOVA, Duncan’s test).

vertical directions (Fig. 2A). The inhibitory rate ranged from 30.3% to flask− 1).
52.9% (Fig. 2B). Apart from F. graminearum, the hyphae of other phy­ The activities of hydrolase in these solutions, including cellulase,
topathogens were twisted, deformed, and or folded in the VCs treatment chitinase, β-glucanase, phosphatases, and protease, also were increased
compared to CK (Fig. 3). after exposure to the phytopathogenic cell wall in comparison to CK (no
significant difference were found in β-glucanase and phosphatases be­
3.2. Mycoparasitism of C. lacerata HG2011 on phytopathogen fungi tween CK and glucose with the addition of the cell wall powder of
phytopathogens, Fig. 6 B–F).
When C. lacerata HG2011 and phytopathogen met each other in the Fungal broth treated with P. capsici cell walls demonstrated higher
dual culture, all test phytopathogens stopped growing (Fig. 4). In activities of cellulose, β-glucanase and protease than those treated with
contrast, the former continued to grow until it covered and lysed the two pathogenic fungal cell walls (A. alternata and D. bryoniae). For
entire colonies of phytopathogens almost after 7-day incubation (Fig. 4, instance, cellulolytic activity showed the highest value of about 2624.8
Fig. S1). Microscopic observation found that C. lacerata HG2011 nKat in P. capsici CW + glucose, followed by 1171.4 nKat in P. capsici
deformed the pathogen hyphae, including twisting, swelling, branching, CW, 997.9 nKat in A. alternata CW, 791.8 nKat in D. bryoniae CW, and
lysing, etc. (Fig. 5) and also lysed and deformed the spore of A. alternata 260.3–390.5 nKat in D. bryoniae CW + glucose, A. alternata CW+ glucose
(Fig. 5EF). and CK. In contrast, chitinase in solution with the presence of two fungal
cell walls was highest. Only protease activity showed a significant cor­
3.3. Growth and hydrolase production of C. lacerata HG2011 in culture relation with mycelia biomass (r = 0.750, n = 28, P < 0.01).
solutions
3.4. Suppression of plant fungal diseases
C. lacerata HG2011 had higher biomass in the culture mediums
containing pathogen cell walls than that in PDB (no significant differ­ Compared with only pathogen inoculation (PI), C. lacerata HG2011
ence in PDB, A. alternata CW and D. bryoniae CW, Fig. 6A). In detail, the inoculation significantly reduced the disease incidence and index of
biomass of C. lacerata HG201 changed in the sequence: P. capsici CW + both eggplant stem blight and cucumber vine blight (Table 1). The
glucose (272.3 mg flask− 1) > A. alternata CW + glucose (238.0 mg control efficacies of C. lacerata HG2011 against cucumber vine blight
flask− 1) > D. bryoniae CW + glucose and P. capsici CW (181.0–196.0 mg and eggplant stem blight range 67.9–70.9%. Chemical pesticides
flask− 1) > A. alternata CW, CK and D. bryoniae CW (111.5–126.25 mg showed higher control efficacies against eggplant stem blight than

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J. Yin et al. Pesticide Biochemistry and Physiology 190 (2023) 105316

Fig. 2. Antagonistic activity of volatile compounds (VCs) emitted by C. lacerata HG2011 against plant pathogens tested in vitro. (A) The colonies of phytopathogens
on PDA exposure with and without VCs emitted by C. lacerata HG2011 after 5-day incubation. (B) Inhibition percentage of phytopathogenic colonies with and
without VCs of C. lacerata HG2011. Bar represents standard error and the distinct small letters indicate significant differences across treatments at P ≤ 0.05 (One-way
ANOVA, Duncan’s test).

C. lacerata HG2011. By contrast, PI+CL and PI+CP achieved similar and inducing plant systemic resistance and so forth (Alabouvette et al.,
control efficacies against cucumber vine blight. 2006). In this study, the crude extract of fungal broth (CEFB) and vol­
atile compounds (VCs) produced by C. lacerata HG2011 greatly sup­
4. Discussion pressed the growth of all tested phytopathogen fungi and oomycetes in
vitro (Figs. 1 and 2). The VCs from this fungus caused phytopathogenic
Taxonomically, Ceriporia lacerata belongs to Basidiomycota, Agar­ hypha to twist, deform and fold, to some extent (Fig. 3). Those results
icomycetes, Polyporales， Irpicaceae. The classification of this fungus is indicated that C. lacerata HG2011 could synthesize multiple antibiotics
still controversial, which is traditionally placed in Ceriporia genus since to suppress pathogenic fungi and oomycetes concurrently by changing
it was discovered and named by 2003 (Suhara et al., 2003) but some mycelial morphology, consistent with those found on other BCMs
scientists recently proposed to transfer it into the genera of Emmia (Wu (Massawe et al., 2018; Sanchez et al., 2019). The co-presence of soluble
et al., 2017) and Irpex (Chen et al., 2021). C. lacerata is known to cause and volatile antimicrobial metabolites indicated that antibiotics pro­
white rot of angiosperm and gymnosperm wood (Suhara et al., 2003). duced by this fungus were with diverse structures and targets, corre­
However, this fungus can live with non-woody plants together as an sponding to other reports about multiple antimicrobial metabolites
endophytic fungus, such as Huperzia serrata (Yu et al., 2018), Cleistocalyx biosynthesized by C. lacerata species (Zhao et al., 2013; Zhang et al.,
operculatus (Wang et al., 2013), Oryza sativa (Lapuz et al., 2018) and 2015; Yu et al., 2018), which could be part of explanations for its good
black pepper (Sreeja et al., 2016), etc. C. lacerata significantly promoted antagonistic effect on all tested phytopathogens. Besides, the antimi­
the growth of Michelia macclurei (Pan et al., 2022), and our early report crobial VCs contribute to C. lacerata HG2011 broad-scope protect plants
demonstrated that C. lacerata HG2011 could also improve the growth via indirect contact and competition with pathogens when they occur in
and yield of several crops like eggplant, tomato and broad bean (Yin rhizosphere, due to the excellent performance of low-molecular-weight
et al., 2021a, 2021b; Yin et al., 2022). To reduce risk in the use of VCs on long-distance migration in soil (Morath et al., 2012). However, it
C. lacerata HG2011, more crops need to be tested and this fungus can be needs to be further investigated the characteristics of these metabolites.
used only on the tested plants to control diseases. In the dual culture, C. lacerata HG2011 exhibited a faster growth rate
The main antagonistic mechanisms of biocontrol microorganisms than the majority of tested phytopathogens apart from P. capsici and all
(BCMs) include antibiosis, mycoparasitism, competition for nutrients pathogens stopped growing once contacting with the former (Fig. 4).

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J. Yin et al. Pesticide Biochemistry and Physiology 190 (2023) 105316

Fig. 3. Changes in the mycelial morphology of phytopathogens treated with VCs emitted from C. lacerata HG2011. (A) F. graminearum, (B) C. gloeosporioides, (C)
R. solani, (D) B. cinerea, (E) D. bryoniae, (F) A. alternata, (G) P. nicotianae, (H) P. capsici; Small letters are the corresponding control (CK), respectively. Bar represents
20 μm. The deformation of phytopathogen hyphae, including expansion, distortion, thickening, increased branches, and lysing, can be visible.

Fig. 4. Dynamic changes of C. lacerata HG2011 and phytopathogens in dual culture.

C. lacerata HG2011 was on the left side and phytopathogens were on the right side in each plate. From A to H were F. graminearum, C. gloeosporioides, R. solani,
B. cinerea, D. bryoniae, A. alternata, P. nicotianae and P. capsici, respectively.

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J. Yin et al. Pesticide Biochemistry and Physiology 190 (2023) 105316

Fig. 5. The hyphal morphology of plant pathogens in the zone contacting with C. lacerata HG2011. From A to E were C. lacerata HG2011 and pathogens in dual
culture (A, D. bryoniae; B, P. capsici; C, P. nicotianae; D, F. graminearum; E, A. alternata). F) the hypha of A. alternata when separately growing on PDB. Bars represent
20 μm.

Early reports revealed that this fungus could release many compounds phytopathogens as an invasive mycoparasite.
(eg. siderophore) and enzymes related to the soil nutrient cycle (Yin One of the most essential mechanisms in attacking the host microbes
et al., 2021b; Yin et al., 2022). The capacity for rapid growth and by necrotrophic mycoparasite is the hydrolases release that are
nutrient acquisition were favorable for C. lacerata HG2011 to compete responsible for lysing phytopathogen cell wall (CW), which is comprised
for resources and space, inhibiting the growth of fractional phytopath­ of 90% wall polysaccharides (β-glucan, cellulose, chitin) and minor
ogen when sharing the same ecological niche. C. lacerata HG2011 components like proteins (Latgé, 2007). Similar to mycoparasite Tri­
gradually covered the colonies of all tested phytopathogens in dual choderma harzianum, C. lacerata HG2011 could directly grow on the
culture (Fig. 4, Fig. S1). Correspondingly, phytopathogen hyphae were surface of the living or dead pathogens colonies (Fig. S2). Mycelium
penetrated, broken, and lysed by C. lacerata HG2011 in the contact zone biomass and hydrolases activities (including cellulase, chitinase, β-glu­
(Fig. 5). These phenomena were consistent with the behavior of canase and protease) of this biocontrol fungus were prominently
necrotrophic mycoparasite like some reputed Trichoderma spp. that were increased with the pathogenic cell wall in comparison to growing in PDB
developed as biocontrol agents due to the broad hosts and powerful medium alone (Fig. 6). Therefore, it is reasonable to presume that
destructive ability to pathogens (Chen et al., 2016; Sanchez et al., 2019). C. lacerata HG2011 could colonize the pathogen colony and utilize the
Necrotrophic mycoparasite kills prey through three typical behaviors, pathogen cell wall as nutrient sources for its growth by the effusion of
including the release of secondary metabolites without any contact (eg. those extracellular enzymes. Interestingly, β-glucanase activity from
Clonostachys rosea), coiling around host hyphae without penetration but only was stimulated in the medium with pathogen CW alone, while the
with haustoria (eg. Arthrobotrys oligospora), and penetration into host activities of fungal cellulase and chitinase were accelerated in pathogen
hyphae through antibiotics and hydrolases (invasion, Sun et al., 2019). CW medium with the presence of exogenous glucose or not. This result
In the current experiment, C. lacerata HG2011 attacked different tested suggested the β-glucanase released by C. lacerata HG2011 in

7
J. Yin et al. Pesticide Biochemistry and Physiology 190 (2023) 105316

Fig. 6. Growth and hydrolase production of C. lacerata HG2011 grown in the culture solutions containing the cell walls of different phytopathogens as nutrient
sources.
CK, P. capsici CW, A. alternata CW, D. bryoniae CW, P. capsici CW + glucose, A. alternata CW + glucose, and D. bryoniae CW + glucose represents normal PDB(0.4%
glucose, control), glucose in PDB replaced with the equal-weight cell wall powder of phytopathogens (P. capsici, A.alternata, and D. bryoniae), and PDB (0.4% glucose)
with the addition of the cell wall powder of phytopathogens (P. capsici, A.alternata, and D. bryoniae) respectively. Values are expressed as means ±SE (n = 4) by one-
way ANOVA. Different small letters represent significant differences among treatments at P ≤ 0.05 (One-way ANOVA, Duncan test).

activities trigged by P. capsici CW and chitinase activities trigged by

Table 1
fungal CW (A. alternata and D. bryoniae), associated with the distinct
Effects of C. lacerata HG2011 on the disease incidence, index, and control effi­
composition of cell wall polysaccharides between fungi and oomycetes
cacy of crops disease in the greenhouses.
which consists of β-glucan, cellulose and hemicellulose mainly in
Crop Treatment Incidence/ Disease Control oomycete (eg. P. capsici) CW and β- glucan and chitin in fungal CW
% index efficacy/%
(Latgé, 2007; Mélida et al., 2013). Most mycoparasitic fungi are ineffi­
Eggplant stem cient to oomycetes and tend to specialize in one or few fungal species
CK 0.0 ± 0.0d 0.0 ± 0.0d –
blight
due to the species-specific effectiveness of their artillery (Rangel et al.,
64.4 ±
PI 67.8 ± 6.9a
7.00a
– 2021). For example, only T. harzianum and Trichoderma hamatum among
PI+ CL 23.3 ± 3.3b 20.7 ± 2.4b 67.9 ± 3.7b tested Trichoderma species successfully degraded the cell wall of
PI+CFC 6.7 ± 3.3c 5.6 ± 2.7c 92.8 ± 4.5a oomycete Pythium ultimum (Inglis and Kawchuk, 2002), while another
Cucumber vine broad host mycoparasite C. rosea and specific mycoparasite Escovopsis
CK 0.0 ± 0.0d 0.0 ± 0.0c –
blight
PI 36.7 ± 3.3a 36.7 ± 3.1a –
weberi lost this ability due to lack of cellobiohydrolases and poly­
PI +CL 16.7 ± 6.7b 10.9 ± 3.7b 70.9 ± 10.01a saccharide monooxygenases auxiliary proteins (de Man et al., 2016).
PI+ CFC 21.1 ± 1.9b 18.7 ± 6.0b 56.36 ± 8.2a C. lacerata HG2011 can produce extracellular β-glucanase, cellulase and,
CK = seedling alone, PI = phytopathogens inoculation, PI+ CL = C. lacerata chitinase and so, by which discharge corresponding substrates leading to
inoculation, CFC = chemical fungicide. Values are expressed by means ±SE (n = mycoparasitism with oomycetes and multiple pathogen fungi (Verma
3) by one-way ANOVA. In each column, means followed by distinct small letters et al., 2007). In support of the phenomena, the genes encoding β-glu­
within the same crop were significantly different at P ≤ 0.05 (Duncan test). canase and cellulose were cloned from C. lacerata rather than C. rosea, E.
weberi, and some Trichoderma species (Karlsson et al., 2017; Mao et al.,
mycoparasitism was induced by carbon competition and the cellulase 2022). The effectively antagonistic effect mediated by secondary me­
and chitinase specifically responded to pathogen CW, similar to the tabolites (particularly VCs) from C. lacerata HG2011 on phytopathogens
mechanism of biocontrol yeast acted on B. cinerea and Penicillium also contributed to the early mycoparasitic interaction between this
expansum were reported (Castoria et al., 1997). Higher cellulase fungus and phytopathogens, which created favorable conditions for

8
J. Yin et al. Pesticide Biochemistry and Physiology 190 (2023) 105316

C. lacerata HG2011 to attack the deformed hyphal and growth-inhibited Author’s contribution
colonies.
The content of β-glucan is much above the level of chitin or cellulose, Conceptualization: Jie Y, Ling Y, Jianguo H. Investigation: Jie Y,
accounting for about 90% of wall polysaccharides for almost fungi and Ruxia B. Writing – Original Draft: Jie Y and Jianguo H. Writing – Review
oomycetes (Geoghegan et al., 2017). β-glucanase activity in all & Editing: Jie Y, Ling Y, Jianguo H. Funding Acquisition: Ling Y, Jianguo
C. lacerata HG2011 broth was much higher (about 8–30 times) than H.
other lytic enzymes. Protease showed a positive correlation with
biomass, indicating this fungus could persistently produce protease and Declaration of Competing Interest
act on pathogens when successfully surviving in the rhizosphere.
Considering the importance of wall β-glucan and protein for cell wall No conflict of interest exists in this manuscript submitted.
architecture, membrane-bound sensor proteins and infect recognition of
pathogens to host plants (Geoghegan et al., 2017), the role of β-gluca­ Data availability
nases and protease in the mycoparasitic process of this fungus with
tested phytopathogen could also not be ignored. Similar abiotic results Data will be made available on request.
have also been reported that the combination of the purified β-gluca­
nases and chitinase resulted in a stronger antagonistic effect on patho­ Acknowledgments
gens in comparison to the single enzyme used (Boller, 1993; Giannakis
et al., 1998). Higher cellulose, β-glucanase and protease in solution with This work was supported by the Chongqing Science and Technology
P. capsici CW were found than those with tested fungal CW, indicating Commission, China (Projects cstc2017shms-xdny80084 and
that C. lacerata HG2011 could be more easily hydrolyzed and utilize cstc2018jscx-mszdX0011). We would like to be grateful to anonymous
oocyte cell walls than fungal’s, which could provide part of explanations referees for their helpful comments on the manuscript.
for the results that higher mycelial biomass was found in medium
exposure to P. capsici CW and markedly faster growth of C. lacerata
Appendix A. Supplementary data
HG2011 on live P. capsici colonies than that on pathogenic fungus
(Fig. 6A, Fig. S2).
Supplementary data to this article can be found online at https://doi.
Although C. lacerata HG2011 was effective in inhibiting the patho­
org/10.1016/j.pestbp.2022.105316.
gens in vitro, controlling the disease caused by these pathogens is
another story. Thus, we further evaluated the biocontrol efficacy of this
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