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Citation: Si, B.; Wang, H.; Bai, J.;
Zhang, Y.; Cao, Y. Evaluating the
Utility of Simplicillium lanosoniveum, a
Hyperparasitic Fungus of Puccinia
graminis f. sp. tritici, as a Biological
Control Agent against Wheat Stem
Rust. Pathogens 2023,12, 22.
https://doi.org/10.3390/
pathogens12010022
Academic Editor: Tomasz Kulik
Received: 18 November 2022
Revised: 16 December 2022
Accepted: 19 December 2022
Published: 23 December 2022
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
pathogens
Article
Evaluating the Utility of Simplicillium lanosoniveum,
a Hyperparasitic Fungus of Puccinia graminis f. sp. tritici, as
a Biological Control Agent against Wheat Stem Rust
Binbin Si 1,2 , Hui Wang 2, Jiaming Bai 2, Yuzhen Zhang 2and Yuanyin Cao 1,*
1College of Plant Protection, Shenyang Agricultural University, Shenyang 110866, China
2College of Biological Science and Engineering, North Minzu University, Yinchuan 750021, China
*Correspondence: caoyy6655@163.com; Tel.: +86-136-0400-0358
Abstract:
Wheat stem rust is one of the wheat diseases caused by Puccinia graminis Pers. f. sp.
tritici (Pgt). This disease has been responsible for major losses to wheat production worldwide.
Currently used methods for controlling this disease include fungicides, the breeding of stem rust-
resistant cultivars, and preventive agricultural measures. However, the excessive use of fungicides
can have various deleterious effects on the environment. A hyperparasitic fungus with white
mycelia and oval conidia,
Simplicillium lanosoniveum
, was isolated from the urediniospores of Pgt.
When Pgt-infected wheat leaves were inoculation with isolates of S. lanosoniveum, it was found that
S. lanosoniveum
inoculation inhibited the production and germination of urediniospores, suggesting
that
S. lanosoniveum
could inhibit the growth and spread of Pgt. Scanning electron microscopy
revealed that S. lanosoniveum could inactivate the urediniospores by inducing structural damage.
Overall, findings indicate that S. lanosoniveum might provide an effective biological agent for the
control of Pgt.
Keywords:
wheat stem rust; Puccinia graminis f. sp. tritici; hyperparasitism; Simplicillium lanosoniveum;
identification
1. Introduction
Eight major cereal crops, wheat, rice, barley, rye, oats, corn, sorghum, and millet,
provide two-thirds of the world’s food supply. Each of these major cereal crops is vulnerable
to at least one rust disease. Rust pathogens can induce substantial damage to cereal crops,
reduce yields, and cause major economic losses [
1
]. Wheat stem rust poses a major threat to
wheat production worldwide because outbreaks of this disease can occur over large spatial
scales and spread rapidly [
2
]. Wheat stem rust infections affect the stems and leaves of
wheat plants. In severe cases, wheat stem rust can reduce wheat yield by 75% or result in
the death of host plants [3].
Hyperparasitic fungi parasitize other plant pathogens, and both hyperparasitic fungi
and their hosts impose mutual constraints on each other’s growth [
4
]. Hyperparasitic
fungi are widespread in natural ecosystems and commonly parasitize filamentous fungi [
5
].
These fungi might provide promising alternatives to chemical fungicides for the control
of fungal diseases in plants [
6
]. They have thus received much research attention from
botanists for their potential to be used as biocontrol agents against plant pathogens. To date,
hyperparasitic fungi have been used to control a few types of rust diseases and powdery
mildew [
7
]. Following the colonization of host pathogens, the hyphae of hyperparasitic
fungi grow toward the edge of the colony. Once the hyphae infiltrate the cell wall of the host
to obtain nutrients, a parasitic relationship is established between the hyperparasitic fungus
and the host pathogen, which can induce damage to the pathogen. Many hyperparasitic
fungi occur naturally in plants in the form of mold colonies.
Pathogens 2023,12, 22. https://doi.org/10.3390/pathogens12010022 https://www.mdpi.com/journal/pathogens
Pathogens 2023,12, 22 2 of 9
Hyperparasitic fungi effectively prevent plant pathogens from infecting their hosts;
these interactions between the plant pathogen and hyperparasite can inhibit the reproduc-
tive ability of the pathogenic organism [
8
,
9
]. The antagonism between plant and parasitic
fungi and that between parasitic fungi and their hyperparasitic fungi can inhibit the growth
and reproductive capacity of pathogenic organisms and thus reduce the severity of plant
diseases [
8
]. Rapidly growing hyperparasitic fungi might have a lower impact on the
environment compared with chemical fungicides, given that they are ubiquitous in natural
ecosystems. This observation has inspired much interest in the development of strategies
to use these fungi as biological control agents [9].
Many biological antagonists have been shown to work effectively against rust fungi.
The application of hyperparasitic fungi is potentially an effective strategy to prevent
plant diseases. A total of 30 hyperparasitic fungal genera parasitize rust fungi, including
Fusarium (Fusarium spp.), Cladosporium,Scytalidium uredinicola, and Tuberculina [
10
,
11
]. Par-
asitism of the urediniospores of rust fungi has been reported in various fungal species [
12
],
including C. cladosporioides,Eudarluca caricis,Microdochium caricis,Microdochium nivale,
Lecanicillium lecanii, and Alternaria alternata. Several Cladosporium species, such as C. uredini-
cola,C. cladosporioides,C. pseudocladosporioides, and C. sphaerospermum, have been reported
to parasitize fungi in the order Pucciniales [13].
Scanning electron microscopy (SEM) of the abnormal urediniospores of wheat stripe
rust revealed that they were parasitized by Cladosporium spp., which inactivated the ure-
diniospores and induced structural damage [
14
]. Multiple hyperparasitic fungi have been
isolated from infected branches of Pinus armandii, and the defense mechanisms of fungi
against Cronartium ribicola have been characterized using SEM [
15
]. The hyperparasitic
fungus Alternaria alternata was first reported to parasitize wheat stripe rust in 2017, and its
potential utility as a biocontrol agent was demonstrated in a previous study [5].
Current methods used for the control of stem rust include the application of fungicides,
the use of stem rust-resistant varieties, and preventive agricultural measures [
16
]. However,
the application of nonselective fungicides can lead to a disease more difficult to manage [
9
].
In addition, the heavy use of fungicides can have deleterious effects on the environment,
along with animals and humans [
6
]. Here, the utility of hyperparasitic fungi for the control
of Pgt infection is evaluated. Our findings indicate that hyperparasitic fungi could provide
an effective tool for controlling destructive rust diseases of major cereal crops. The isolates
of the hyperparasitic fungi in our experiment effectively inhibit the growth of Pgt and will
aid future studies aimed at the development of environmentally friendly biological agents.
2. Materials and Methods
2.1. Isolation and Purification of Hyperparasitic Fungi
We were multiplying Pgt urediniospores and conducting various experiments with
urediniospores in our laboratories at Shenyang Agricultural University, Shenyang. Hy-
perparasitic fungi were isolated from the urediniospores of Pgt, which were cultured at
22
◦
C and 60% relative humidity in an artificial climate chamber. The susceptible wheat
cultivar Little Club was inoculated with Pgt (34C3RTGQM) and then placed in an artificial
climate chamber, with 60% relative humidity, temperatures kept at 22
◦
C, and a 16 h/8 h
light/dark photoperiod.
The white hyphae were collected from the urediniospores of Pgt-infected wheat leaves,
inoculated onto potato dextrose agar (PDA) plates using an inoculation loop following
16 d
of culture, and then incubated in an inverted position at 25
◦
C for 7–10 d in a biochemical
incubator. After extracting fresh hyphae from the edge of the fungal colony, they were
transferred to fresh sterile PDA medium and incubated under the same conditions to obtain
a pure culture. Five replications of these experiments were performed. The purified isolates
of hyperparasitic fungus were preserved on the PDA plate.
Pathogens 2023,12, 22 3 of 9
2.2. Morphological Observations of Hyperparasitic Fungi
Hyphae with vigorous growth at the edge of the isolated and purified colonies were
transferred to fresh sterile PDA medium and placed in an incubator with a 12 h/12 h
dark/light photoperiod at 25
◦
C for 7 d to make observations of the morphology of its
colonies, hyphae, and conidia. Observations of hyphal and conidial morphology were
performed using an Olympus BX51 microscope.
2.3. SEM of Stem Rust Fungi and Hyperparasitic Fungi
Leaves inoculated with only Pgt urediniospores were used as the CK. The hyperpara-
sitic of isolate fungus was placed into a fungal spores suspension (10
5
–10
6
/mL), which was
sprayed on the Pgt-infected wheat leaves. One day after Pgt inoculation, the Pgt-infected
leaves were inoculated with a suspension of isolate fungus, and kept in the artificial cli-
mate chamber for growth under the same conditions. To clarify Pgt–parasite interactions
during infection, the Pgt-infected wheat leaves of hyperparasitic were cut into blocks
(
0.2 cm ×0.2 cm
) and stored in fixative for SEM. The leaves were sampled at 0, 1, 3, 5, 7,
and 9 d after smearing the hyperparasitic fungal suspension.
2.4. Molecular Identification of Hyperparasitic Fungi
Purified hyphae (0.1 g) of hyperparasitic fungus were placed into 2.0 mL centrifuge
tubes. The OMEGA (USA) HP Fungal DNA Kit was used to extract DNA from samples.
A NanoDrop Microvolume Spectrophotometer (Thermo) was used to determine the con-
centration of DNA. Polymerase chain reaction (PCR) was then performed to amplify the
internal transcribed spacer 1 (ITS1) and internal transcribed spacer 4 (ITS4) sequences (ITS1
primer: 5
0
-TCCGTAGGTGAACCTGCG-3
0
; ITS4 primer: 5
0
-TCCTCCGCTTATTGATATGC-
3
0
) [
17
,
18
]. Electrophoresis with a 1.0% agarose gel was performed to analyze the PCR-
amplified products. The thermal cycling conditions were as follows: denaturing at 94
◦
C
for 5 min; 30 cycles (from 94
◦
C for 35 s, 52
◦
C for 60 s, to extension at 72
◦
C for 90 s); to
extension at 72
◦
C for 10 min; and 4
◦
C for 5 min. PCR products were separated by 1% gel
electrophoresis. The extracted DNA samples of the hyperparasitic fungi of Pgt were sent to
Sangon Biotech Co. (Shanghai, China) for sequencing and molecular identification.
2.5. Phylogenetic Analysis
The sequences of Simpliciium spp. were downloaded from the NCBI and aligned
by ClustalW in software MEGA10 using the default parameters [
19
]. The fungal isolates
were clustered based on their ITS sequences using the neighbor-joining (NJ) method using
MEGA10, and the branch robustness was determined using 1000 bootstrap replications [
19
].
2.6. Hyperparasitic Fungal Inhibition Experiment
The susceptible wheat (Little Club) was used for propagating Pgt urediniospores.
When the first leaf had expanded after 7 days, seedlings were inoculated with 34C3RTGQM,
a predominant race of Pgt in China. The collected urediniospores of Pgt race 34C3RTGQM
were diluted with water to 25 mg
·
mL
−1
and inoculated by brush. The Pgt-inoculated
plants were incubated in an artificial climate chamber at 22
◦
C with 16 h light photoperiod.
The hyperparasitic of fungus was placed into a fungal spores suspension (10
5
–10
6
/mL),
which was sprayed on the Pgt-infected wheat leaves. Healthy wheat leaves receiving
S. lanosoniveum
inoculation represented control check1 (CK1). Leaves inoculated with only
Pgt urediniospores were used as CK2. Three, six, and nine days after Pgt inoculation, the
plants in different pots were inoculated with suspension (10
5
~10
6
spores/mL) of hyper-
parasite, and kept in the artificial climate chamber for growth under the same conditions.
Each treatment was carried out with wheat seedlings growing in three independent pots.
All treatments were placed in the same artificial climate chamber, and observation of the
symptoms was performed at the same time.
Pathogens 2023,12, 22 4 of 9
2.7. Urediniospore Germination Inhibition Experiment
Seedlings of wheat (Little Club) were grown in the artificial climate chamber, wait-
ing until the first leaf had expanded after 7 days, inoculated with urediniospores of Pgt
(34C3RTGQM), and incubated in an artificial climate chamber at 22
◦
C. Fungal suspen-
sions (10
5
–10
6
CFU/mL) were prepared in sterile water with fresh urediniospores and
hyperparasitic fungal hyphae, which were extracted with a sterilized inoculating loop. The
treatment group comprised a mixture of equal volumes of urediniospore suspension and
hyphal suspension. In the control group, the urediniospore suspension was mixed with
equal volumes of sterile water and cultured in a biochemical incubator in the dark at 22
◦
C
for 6 h in an artificial climate chamber. Suspensions from the treatment and control groups
were transferred to glass slides to perform counts of the germinated urediniospores using a
light microscope. Germinating urediniospores of Pgt produce a germ tube that is at least
half the length of the urediniospores. The germination rate was expressed as a percentage
based on 100 randomly selected urediniospores. Experiments were performed in triplicate.
3. Results and Analysis
3.1. Morphological Observations of Hyperparasitic Fungi
The hyperparasitic fungus was white and slow-growing on the PDA medium
(
Figure 1A
). Following 10 d of incubation at 25
◦
C, fungal colonies (50–60 mm in di-
ameter) were dense and appeared brownish-orange on the bottom of the dish (Figure 1B).
Microscopic observation revealed intertwined hyphae variable in diameter, oval conidia,
and spores that were stacked or arranged in rows. The hyphae and conidia of the isolated
filamentous fungus were highly similar to those of Simplicillium spp.
Pathogens 2022, 11, x FOR PEER REVIEW 4 of 10
ter Pgt inoculation, the plants in different pots were inoculated with suspension (10
5
~10
6
spores/mL) of hyperparasite, and kept in the artificial climate chamber for growth under
the same conditions. Each treatment was carried out with wheat seedlings growing in
three independent pots. All treatments were placed in the same artificial climate cham-
ber, and observation of the symptoms was performed at the same time.
2.7. Urediniospore Germination Inhibition Experiment
Seedlings of wheat (Little Club) were grown in the artificial climate chamber, wait-
ing until the first leaf had expanded after 7 days, inoculated with urediniospores of Pgt
(34C3RTGQM), and incubated in an artificial climate chamber at 22 °C. Fungal suspen-
sions (10
5
–10
6
CFU/mL) were prepared in sterile water with fresh urediniospores and
hyperparasitic fungal hyphae, which were extracted with a sterilized inoculating loop.
The treatment group comprised a mixture of equal volumes of urediniospore suspension
and hyphal suspension. In the control group, the urediniospore suspension was mixed
with equal volumes of sterile water and cultured in a biochemical incubator in the dark
at 22 °C for 6 h in an artificial climate chamber. Suspensions from the treatment and
control groups were transferred to glass slides to perform counts of the germinated ure-
diniospores using a light microscope. Germinating urediniospores of Pgt produce a
germ tube that is at least half the length of the urediniospores. The germination rate was
expressed as a percentage based on 100 randomly selected urediniospores. Experiments
were performed in triplicate.
3. Results and Analysis
3.1. Morphological Observations of Hyperparasitic Fungi
The hyperparasitic fungus was white and slow-growing on the PDA medium (Fig-
ure 1A). Following 10 d of incubation at 25 °C, fungal colonies (50–60 mm in diameter)
were dense and appeared brownish-orange on the bottom of the dish (Figure 1B). Mi-
croscopic observation revealed intertwined hyphae variable in diameter, oval conidia,
and spores that were stacked or arranged in rows. The hyphae and conidia of the isolat-
ed filamentous fungus were highly similar to those of Simplicillium spp.
Figure 1. A and B show the morphology of the spores and hyphae of the hyperparasitic fungus on
the top and bottom of the dish; C and D show the characteristics of hyphae and conidia in mor-
phology under a scanning electron microscope (×500, ×2000).
3.2. Molecular Identification of Hyperparasitic Fungi
Figure 1.
(
A
,
B
) show the morphology of the spores and hyphae of the hyperparasitic fungus on the
top and bottom of the dish; (
C
,
D
) show the characteristics of hyphae and conidia in morphology
under a scanning electron microscope (×500, ×2000).
3.2. Molecular Identification of Hyperparasitic Fungi
The hyperparasitic strain IS698-2 was isolated from Pgt urediniospores. This hyper-
parasitic strain of IS698-2 was sequenced and identified as Simplicillium lanosoniveum.
Phylogenetic analysis was performed on Simplicillium. Alignments of the gene se-
quences were performed using data and tools available in the National Center for Biotech-
nology Information database. The phylogeny of fungal ITS sequences was built using the
neighbor-joining (NJ) method with 1000 bootstrap replicates in MEGA10software (Figure 2).
Pathogens 2023,12, 22 5 of 9
Pathogens 2022, 11, x FOR PEER REVIEW 5 of 10
The hyperparasitic strain IS698-2 was isolated from Pgt urediniospores. This hy-
perparasitic strain of IS698-2 was sequenced and identified as Simplicillium lanosoni-
veum.
Phylogenetic analysis was performed on Simplicillium. Alignments of the gene se-
quences were performed using data and tools available in the National Center for Bio-
technology Information database. The phylogeny of fungal ITS sequences was built us-
ing the neighbor-joining (NJ) method with 1,000 bootstrap replicates in
MEGA10software (Figure 2).
Figure 2. Phylogenetic tree of Simplicillium constructed using the NJ method. The red dot indi-
cates the hyperparasitic S. lanosoniveum isolated in this study.
3.3. Pathogenicity Test to Confirm Hyperparasitism: S. lanosoniveum Parasitizing Pgt
The hyperparasitic ability of S. lanosoniveum was confirmed by pathogenicity testing
(Figure 3). Fungal suspensions of S. lanosoniveum with spore concentrations of 10
5
–10
6
/mL were evenly sprayed on wheat leaves without any symptoms (Figure 3A). Leaves
inoculated with only Pgt urediniospores produced abundant urediniospores after 12
days post-inoculation (Figure 3B, C). The suspensions of S. lanosoniveum inoculated with
Pgt-infected wheat leaves were observed at 3, 6, and 9 dpi (Figure 3D, E, F); 3 and 6 dpi
were without any symptoms (Figure 3D, E), but at 9 dpi, white hyphae wrapped the
pustule (Figure 3F).
The production of urediniospores ultimately ceased, suggesting that S. lanosoni-
veum inhibited the urediniospore production of Pgt. This also suggests a possible para-
sitic relationship between S. lanosoniveum and Pgt.
Figure 2.
Phylogenetic tree of Simplicillium constructed using the NJ method. The red dot indicates
the hyperparasitic S. lanosoniveum isolated in this study.
3.3. Pathogenicity Test to Confirm Hyperparasitism: S. lanosoniveum Parasitizing Pgt
The hyperparasitic ability of S. lanosoniveum was confirmed by pathogenicity testing
(Figure 3). Fungal suspensions of S. lanosoniveum with spore concentrations of 10
5
–10
6
/mL
were evenly sprayed on wheat leaves without any symptoms (Figure 3A). Leaves inoc-
ulated with only Pgt urediniospores produced abundant urediniospores after 12 days
post-inoculation (Figure 3B, C). The suspensions of S. lanosoniveum inoculated with Pgt-
infected wheat leaves were observed at 3, 6, and 9 dpi (Figure 3D, E, F); 3 and 6 dpi were
without any symptoms (Figure 3D, E), but at 9 dpi, white hyphae wrapped the pustule
(Figure 3F).
Pathogens 2022, 11, x FOR PEER REVIEW 6 of 10
Figure 3. The pathogenicity test to confirm that S. lanosoniveum could hyperparasitize Pgt. A: CK1, wheat
leaves (cultivar: Little Club) inoculated with S. lanosoniveum, 12 dpi, without symptoms; B, C: CK2,
symptoms on wheat leaves (Little Club) inoculated only with Pgt, 12 dpi; D, E: wheat leaves
sprayed with S. lanosoniveum suspension following inoculation with Pgt at 3 and 6 dpi. No ure-
diniospores were observed on the leaves at 12 dpi; F: pustules on wheat leaves inoculated with Pgt
at 9 dpi (same cultivar and race as above), and white hyphae wrapped pustules inoculated at 15
dpi with S. lanosoniveum suspension.
3.4. Urediniospore Production Inhibition Experiment
The experiment was performed in triplicate, and average values were used in the
analysis. The germination rate of urediniospores in the treatment group was 17% (17
germinated urediniospores out of 100 total urediniospores examined); the germination
rate in the control group was 91% (i.e., 91 germinated urediniospores out of 100 total
urediniospores) (Figures 4 and 5). These findings demonstrated that S. lanosoniveum can
inhibit the germination of Pgt urediniospores.
Figure 4. A shows Pgt urediniospores cultures in water; B shows the cultures of Pgt uredini-
ospores and S. lanosoniveum in water.
Figure 3.
The pathogenicity test to confirm that S. lanosoniveum could hyperparasitize Pgt. (
A
): CK1, wheat
leaves (cultivar: Little Club) inoculated with S. lanosoniveum, 12 dpi, without symptoms; (
B,C
): CK2,
symptoms on wheat leaves (Little Club) inoculated only with Pgt, 12 dpi; (
D,E
): wheat leaves sprayed
with S. lanosoniveum suspension following inoculation with Pgt at 3 and 6 dpi. No urediniospores
were observed on the leaves at 12 dpi; (
F
): pustules on wheat leaves inoculated with Pgt at 9 dpi
(same cultivar and race as above), and white hyphae wrapped pustules inoculated at 15 dpi with S.
lanosoniveum suspension.
The production of urediniospores ultimately ceased, suggesting that S. lanosoniveum
inhibited the urediniospore production of Pgt. This also suggests a possible parasitic
relationship between S. lanosoniveum and Pgt.
Pathogens 2023,12, 22 6 of 9
3.4. Urediniospore Production Inhibition Experiment
The experiment was performed in triplicate, and average values were used in the analy-
sis. The germination rate of urediniospores in the treatment group was 17% (
17 germinated
urediniospores out of 100 total urediniospores examined); the germination rate in the
control group was 91% (i.e., 91 germinated urediniospores out of 100 total urediniospores)
(Figures 4and 5). These findings demonstrated that S. lanosoniveum can inhibit the germi-
nation of Pgt urediniospores.
Pathogens 2022, 11, x FOR PEER REVIEW 6 of 10
Figure 3. The pathogenicity test to confirm that S. lanosoniveum could hyperparasitize Pgt. A: CK1, wheat
leaves (cultivar: Little Club) inoculated with S. lanosoniveum, 12 dpi, without symptoms; B, C: CK2,
symptoms on wheat leaves (Little Club) inoculated only with Pgt, 12 dpi; D, E: wheat leaves
sprayed with S. lanosoniveum suspension following inoculation with Pgt at 3 and 6 dpi. No ure-
diniospores were observed on the leaves at 12 dpi; F: pustules on wheat leaves inoculated with Pgt
at 9 dpi (same cultivar and race as above), and white hyphae wrapped pustules inoculated at 15
dpi with S. lanosoniveum suspension.
3.4. Urediniospore Production Inhibition Experiment
The experiment was performed in triplicate, and average values were used in the
analysis. The germination rate of urediniospores in the treatment group was 17% (17
germinated urediniospores out of 100 total urediniospores examined); the germination
rate in the control group was 91% (i.e., 91 germinated urediniospores out of 100 total
urediniospores) (Figures 4 and 5). These findings demonstrated that S. lanosoniveum can
inhibit the germination of Pgt urediniospores.
Figure 4. A shows Pgt urediniospores cultures in water; B shows the cultures of Pgt uredini-
ospores and S. lanosoniveum in water.
Figure 4.
(
A
) shows Pgt urediniospores cultures in water; (
B
) shows the cultures of Pgt urediniospores
and S. lanosoniveum in water.
Pathogens 2022, 11, x FOR PEER REVIEW 7 of 10
Figure 5. The germination rate of the mixed culture of hyperparasitic fungi (S. lanosoniveum) and
Pgt urediniospores, and the germination rate of Pgt urediniospores in water. Data are the means of
three independent replicates.
3.5. SEM of Stem Rust Fungi and Hyperparasitic Fungi
SEM was used to study the interaction between S. lanosoniveum and the uredini-
ospores of Pgt. Significant morphological changes in the spores and hyphae of S. lano-
soniveum-parasitized Pgt were observed; specifically: conidia were oval, and multiple
spores were stacked or in groups (Figure 6D, E). Under normal conditions, the uredini-
ospores of Pgt were obovoid or oval (Figure 6A, B). During the initial stage of infection
by hyperparasitic fungi, Pgt urediniospores showed few signs of parasitization (Figure
6C, D). The attachment of S. lanosoniveum spores to urediniospores was observed during
the middle stage of infection (Figure 6E). During the final stage of infection, the uredini-
ospores were completely colonized by hyphae and spores of S. lanosoniveum (Figure 6F).
Changes in the morphology of Pgt urediniospores suggested that S. lanosoniveum
parasitizes Pgt during the sporulation stage, which inactivates the urediniospores. The
spores of S. lanosoniveum began to germinate after contact was made with the uredini-
ospore surface, and the urediniospores of Pgt. were penetrated with its hyphal germ
tube. Following the invasion and colonization of urediniospores, hyperparasites likely
absorbed nutrients from urediniospores, which decreased the viability of urediniospores
and caused them to shrink. The hyphal and conidial morphology of this hyperparasitic
fungus was similar to that of members of the genus Simplicillium.
Figure 5.
The germination rate of the mixed culture of hyperparasitic fungi (S. lanosoniveum) and Pgt
urediniospores, and the germination rate of Pgt urediniospores in water. Data are the means of three
independent replicates. *** p≤0.001.
3.5. SEM of Stem Rust Fungi and Hyperparasitic Fungi
SEM was used to study the interaction between S. lanosoniveum and the urediniospores
of Pgt. Significant morphological changes in the spores and hyphae of S. lanosoniveum-
parasitized Pgt were observed; specifically: conidia were oval, and multiple spores were
stacked or in groups (Figure 6D,E). Under normal conditions, the urediniospores of Pgt
were obovoid or oval (Figure 6A,B). During the initial stage of infection by hyperparasitic
fungi, Pgt urediniospores showed few signs of parasitization (Figure 6C,D). The attachment
of S. lanosoniveum spores to urediniospores was observed during the middle stage of
infection (Figure 6E). During the final stage of infection, the urediniospores were completely
colonized by hyphae and spores of S. lanosoniveum (Figure 6F).
Changes in the morphology of Pgt urediniospores suggested that S. lanosoniveum para-
sitizes Pgt during the sporulation stage, which inactivates the urediniospores. The spores of
S. lanosoniveum began to germinate after contact was made with the urediniospore surface,
and the urediniospores of Pgt. were penetrated with its hyphal germ tube. Following the
invasion and colonization of urediniospores, hyperparasites likely absorbed nutrients from
urediniospores, which decreased the viability of urediniospores and caused them to shrink.
Pathogens 2023,12, 22 7 of 9
The hyphal and conidial morphology of this hyperparasitic fungus was similar to that of
members of the genus Simplicillium.
Pathogens 2022, 11, x FOR PEER REVIEW 7 of 10
Figure 5. The germination rate of the mixed culture of hyperparasitic fungi (S. lanosoniveum) and
Pgt urediniospores, and the germination rate of Pgt urediniospores in water. Data are the means of
three independent replicates.
3.5. SEM of Stem Rust Fungi and Hyperparasitic Fungi
SEM was used to study the interaction between S. lanosoniveum and the uredini-
ospores of Pgt. Significant morphological changes in the spores and hyphae of S. lano-
soniveum-parasitized Pgt were observed; specifically: conidia were oval, and multiple
spores were stacked or in groups (Figure 6D, E). Under normal conditions, the uredini-
ospores of Pgt were obovoid or oval (Figure 6A, B). During the initial stage of infection
by hyperparasitic fungi, Pgt urediniospores showed few signs of parasitization (Figure
6C, D). The attachment of S. lanosoniveum spores to urediniospores was observed during
the middle stage of infection (Figure 6E). During the final stage of infection, the uredini-
ospores were completely colonized by hyphae and spores of S. lanosoniveum (Figure 6F).
Changes in the morphology of Pgt urediniospores suggested that S. lanosoniveum
parasitizes Pgt during the sporulation stage, which inactivates the urediniospores. The
spores of S. lanosoniveum began to germinate after contact was made with the uredini-
ospore surface, and the urediniospores of Pgt. were penetrated with its hyphal germ
tube. Following the invasion and colonization of urediniospores, hyperparasites likely
absorbed nutrients from urediniospores, which decreased the viability of urediniospores
and caused them to shrink. The hyphal and conidial morphology of this hyperparasitic
fungus was similar to that of members of the genus Simplicillium.
Figure 6.
SEM of Pgt-infected wheat leaves infected with hyperparasitic fungi (S. lanosoniveum).
(
A
). CK, wheat leaves (Little Club) inoculated only with Pgt urediniospores. (
B
). Hyphal growth
of hyperparasitic fungi observed on the surface of urediniospores of Pgt at 1 dpi. (
C
). Germ tubes
formed in the hyperparasitic fungi at 3 dpi. (
D
). Pgt urediniospores at the middle stage (5 dpi) of
infection. (
E
). Pgt urediniospores partly covered by hyperparasitic fungal spores at 7 dpi. (
F
). The
complete colonization of urediniospores of Pgt by hyperparasitic fungi at 9 dpi.
4. Discussion
Studies on the parasitism of cereal pathogenic fungi by other fungi are important for
the development of biological agents for the control of diseases in economically important
cereal crops. The identification of hyperparasitic fungi is critically important for understand-
ing their biodiversity. Several hyperparasitic fungi that can parasitize plant rust pathogens
have been reported in previous studies, such as Aphanocladium album, Fusarium spp., Lecani-
cillium spp., and Scytalidium uredinicola [
9
]. Fungal parasitism has been suggested to be an
effective strategy for the control of several diseases. For example, Ampelomyces quisqualis
has been used to control powdery mildew on grapes and other crops [
20
]. Trichoderma
spp. have been used to mitigate the effects of Fusarium oxysporum on tomato plants [
21
].
However, few studies have examined the efficacy of using hyperparasitic fungi for the
control of Pgt on cereal crops. In our study, we isolated a fungus that could parasitize the
urediniospores of Pgt. This hyperparasitic fungus could thus potentially be used for the
control of Pgt, given that it inhibits the development of the urediniospores of Pgt.
We studied the morphology of this hyperparasitic fungus, obtained ITS sequences,
and built a phylogeny using the NJ method in MEGA10. The molecular data indicated that
this fungus belonged to the genus Simplicillium, and the identification of this fungus as
S. lanosoniveum had 99% bootstrap support [22,23].
The most practical approaches for controlling wheat stem rust that are currently used
include the breeding of stem rust-resistant varieties and the application of fungicides. The
loss of fungal-specific resistance in wheat varieties and the potential resistance of pathogens
to fungicides are some of the major challenges in the development of biological agents for
the control of wheat stem rust.
Pathogens 2023,12, 22 8 of 9
5. Conclusions
The present study identified S. lanosoniveum as a new hyperparasite of Pgt. Charac-
terizing the effects of hyperparasitic fungi on plant pathogens is essential for evaluating
their utility as biological agents for the control of plant pathogenic fungi. S. lanosoniveum
inhibited Pgt infection through its inhibitory effects on the development and survival of
the urediniospores of Pgt. However, additional studies are needed to determine whether
S. lanosoniveum
can be used as a biocontrol agent for stem rust under field conditions. More
studies are also needed to evaluate the environmental impact of hyperparasitic fungi and
their potential to be used for the control of other rust pathogens.
Author Contributions:
B.S.: investigation, conceptualization, methodology, data curation, and
writing—original draft. H.W.: investigation and data curation. J.B.: methodology and data curation.
Y.Z.: methodology. Y.C.: resources, validation, and writing—editing. All authors have read and
agreed to the published version of the manuscript.
Funding:
This work was supported by the Fundamental Research Funds for the Central Universities,
North Minzu University (2019KJ22); the Natural Science Foundation of Ningxia (2022AAC03290);
and the Innovation Team for Genetic Improvement of Economic Forests (2022QCXTD04).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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