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Citation: Li, F.; Zhu, R.; Gao, F.; Duan,
T. Prior Infection by Colletotrichum
spinaciae Lowers the Susceptibility to
Infection by Powdery Mildew in
Common Vetch. Plants 2024,13, 52.
https://doi.org/10.3390/
plants13010052
Academic Editors: Livy Williams, Kris
A.G. Wyckhuys, Nicolas Desneux and
Yanhui Lu
Received: 27 November 2023
Revised: 20 December 2023
Accepted: 20 December 2023
Published: 22 December 2023
Copyright: © 2023 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/).
plants
Article
Prior Infection by Colletotrichum spinaciae Lowers the
Susceptibility to Infection by Powdery Mildew in Common Vetch
Faxi Li 1, †, Rui Zhu 1 ,† , Feng Gao 2, * and Tingyu Duan 1,*
1
State Key Laboratory of Herbage Improvement and Grassland Agro-ecosystems, Key Laboratory of Grassland
Livestock Industry Innovation, Ministry of Agriculture and Rural Affairs, College of Pastoral Agriculture
Science and Technology, Lanzhou University, Lanzhou 730000, China; lifx19@lzu.edu.cn (F.L.);
220220902410@lzu.edu.cn (R.Z.)
2Gansu Vocational College of Agriculture, Lanzhou 730020, China
*Correspondence: fenggao74@163.com (F.G.); duanty@lzu.edu.cn (T.D.); Tel.: +86-152-1409-5029 (T.D.)
†These authors contributed equally to this work.
Abstract: Anthracnose (Colletotrichum spinaciae) and powdery mildew (Erysiphe pisi) are important
diseases of common vetch (Vicia sativa) and often co-occur in the same plant. Here, we evaluate how
C. spinaciae infection affects susceptibility to E. pisi, using sterilized and non-sterilized field soil to test
the effect of resident soil microorganisms on the plant’s immune response. Plants infected with C.
spinaciae (C
+
) exhibited a respective 41.77~44.16% and 72.37~75.27% lower incidence and severity of
powdery mildew than uninfected (C
−
) plants. Moreover, the net photosynthetic rate, transpiration
rate, and stomatal conductance were higher in the C
−
plants than in the C
+
plants prior to infection
with powdery mildew. These differences were not recorded following powdery mildew infection.
Additionally, the activities of superoxide dismutase, polyphenol oxidase, and catalase were higher in
the C
+
plants than in the C
−
plants. The resident soil microbiota did not affect the plant responses to
both pathogens. By uncovering the mechanistic basis of plant immune response, our study informs
integrated disease management in a globally important forage crop.
Keywords: common vetch; anthracnose; powdery mildew; reactive oxygen species; defense enzyme
1. Introduction
Common vetch (Vicia sativa) is a member of the Leguminosae family. It originated
from southern Europe and western Asia and is widely cultivated throughout the world [
1
].
Common vetch is an excellent forage crop that is highly resistant to drought, tolerant to
salt, and highly effective at fixing nitrogen. Moreover, it grows quickly and can improve
the productivity of pastures when mixed with other forage grasses [
2
]. Common vetch is a
green manure crop that is commonly used in rotation and intercropping production, such
as intercropping with fruit trees [
3
]. Moreover, the plants have been planted to prevent soil
erosion [4].
Plant diseases significantly affect agricultural production both directly and indirectly
and lead to losses of approximately $40 billion per year worldwide [
5
]. Common vetch
is susceptible to diseases and pathogens, including fungi, bacteria, viruses, and nema-
todes. Approximately 24 fungal diseases, four bacterial diseases, five viral diseases, and
24 nematodes, including southern root-knot nematodes, have been detected in common
vetch [
6
,
7
]. However, anthracnose is one of the most common diseases of common vetch.
The pathogens that cause anthracnose in this plant include Colletotrichum vicia [
8
], C.
villosum [
9
], C. sativum [
10
], C. viciae-sativae [
11
], C. acutatum [
12
], C. lentis [
13
], and C.
spinaciae [
14
]. Colletotrichum species are hemibiotrophic plant pathogenic fungi that feed on
live plant tissues in the early stage of infection and dead cells in the plant residues after
plant death [
15
,
16
]. Anthracnose can cause severe yield losses. For example, it can decrease
the plant biomass by 26.90~40.00%, decrease the numbers of effective nodules by 42.25%,
Plants 2024,13, 52. https://doi.org/10.3390/plants13010052 https://www.mdpi.com/journal/plants
Plants 2024,13, 52 2 of 14
and increase the content of jasmonic acid (JA) by 70.34% under greenhouse conditions [
17
].
Moreover, anthracnose reduces the content of chlorophyll a and b and inhibits the PSII
photochemical activity [
18
]. Colletotrichum spinaciae was detected in Xiahe, Gansu Province,
China, in 2019, and it caused a significant reduction in yield from 2019 to 2021. Anthracnose
can reduce the shoots and seed yield of common vetch by more than 25.8% and 32.5%,
respectively, under field conditions [19].
Powdery mildew is a common fungal disease that affects many plant species, including
the Erysiphe pisi infection of common vetch. This disease decreases the ability to conduct
photosynthesis, which results in severe yield losses [
20
–
22
]. Erysiphe pisi is a type of
biotrophic parasitic pathogenic fungus that coexists with the host and does not cause its
death [
16
,
20
,
23
]. Powdery mildew usually affects the photosynthesis of plants, which leads
to yield losses. For example, powdery mildew can cause approximately 20% yield losses
in winter wheat (Triticum aestivum) [
24
]. Powdery mildew can significantly reduce the
net photosynthesis and evapotranspiration compared to in leaves that are not infected.
Furthermore, the net photosynthesis can be practically interrupted when the infected area
is more than 75% of a young leaf [
25
]. The induction in activities of enzymes related to
defense, such as phenylalanine ammonia lyase, polyphenol oxidase (PPO), and peroxidase
(POD), can decrease the natural incidence of powdery mildew infection by 37.2% [26].
Multiple stresses, such as abiotic ones, diseases, and insect pests, simultaneously affect
crops in the agricultural system. Moreover, many studies have assessed the interaction
between abiotic stress and disease. Most cases have shown that abiotic stress aggravates
diseases. For example, salt and water stresses aggravate the susceptibility of faba bean
(Vicia faba) to phytopathogenic fungi [
27
]. Moreover, banana (Musa nana) and grapevine
(Vitis vinifera) are more vulnerable to diseases and infections under water stress [
28
,
29
]. Fur-
thermore, some studies have analyzed the interactions between pathogens and insects and
found that insect pests may positively or negatively affect the prevalence of diseases [
19
].
In some cases, a pathogenic infection can induce a plant immune response, which renders
the plant resistant to the invasion of other pathogens. For example, infection with Zucchini
yellow mosaic virus (ZYMV) reduces the exposure of a second insect-vectored pathogen
(Erwinia tracheiphila) and the establishment of powdery mildew on Cucurbita pepo [
30
]. Ad-
ditionally, infection with Blumeria graminis can elicit immune responses in wheat (Triticum
aestivum), such as the expression of defense genes (PAL,PR1,PR2,PR3,PEROX, and NAD-
PHOX), the content of total phenolics, and the activity of related antioxidant enzymes, thus
suppressing the development of the English grain aphid (Sitobion avenae) [
31
]. Scientists
have defined these pre-infection interactions as “ecological interference,” which indicates
that a primary infection can reduce the rate of contact and development of secondary
infections [
32
]. The previous infection in plant cells can trigger signaling cascades whose
functions are implemented through the signaling molecules JA and salicylic acid (SA).
The elicited immune system can provide broad-spectrum immunity against subsequent
pathogen attacks [
33
]. However, the interaction between phytopathogenic fungi on forage
crops is unclear.
Microbes in the rhizosphere can protect plants against abiotic and biotic stresses. This
microbial resource has increasing importance for crop production and a reduction in fertil-
izer and pesticide inputs [
34
]. The protection of plants against diseases is one of the most
intensively studied microbiome functions and became well known from the discovery of
disease-suppressive soils [
35
]. For example, infection by the foliar downy mildew pathogen
Hyaloperonospora arabidopsidis promotes the growth of specific microbiota in the rhizosphere
of Arabidopsis thaliana, which indirectly affects the pathogen. These rhizosphere micro-
bial community changes include an enrichment of three beneficial bacteria (Xanthomonas,
Stenotrophomonas, and Microbacterium) that help plants to resist subsequent invasion by
pathogens [36].
Anthracnose and powdery mildew are common diseases of common vetch that sig-
nificantly affect the growth and production of this species in the Qinghai–Tibet Plateau in
China. Infection with anthracnose usually begins in July, while powdery mildew occurs
Plants 2024,13, 52 3 of 14
in September in this plateau. The two diseases simultaneously occur in middle and late
September in the fields of the Qinghai–Tibet Plateau. Therefore, it is necessary to determine
the synergistic effect of the two diseases on common vetch and assess whether anthracnose
affects subsequent infection by powdery mildew. This study was designed to simulate
a field anthracnose infection followed by that of powdery mildew to test the effects of
anthracnose caused by C. spinaciae on a subsequent powdery mildew infection. We estab-
lished different soil microbial treatments through the sterilization and non-sterilization
of field soil with common vetch growth and determined the effects of the resident soil
microorganisms in the field on the response of common vetch to anthracnose and pow-
dery mildew. We proposed the following hypotheses: (1) The synergistic occurrence of
anthracnose and powdery mildew severely damages common vetch, and anthracnose
renders common vetch non-susceptible to powdery mildew, and (2) the resident field soil
microorganisms have a positive effect on the resistance of common vetch to anthracnose
and powdery mildew.
2. Materials and Methods
2.1. Plants and Pathogens
Vicia sativa “Lanjian No. 2” was obtained from Lanzhou University (Lanzhou, China).
This is the primary common vetch cultivar that is recommended by the Ministry of Agri-
culture and Rural Affairs of China and widely planted in the Qinghai–Tibet Plateau. The
anthracnose pathogen was isolated from infected leaves of common vetch in Xiahe County,
Gansu Province. The pathogen was isolated from the diseased common vetch in the field
and identified as C. spinaciae using morphological, molecular biological, and pathogenicity
determination [
14
]. Powdery mildew occurs naturally in the individual plant pathology
greenhouse.
Experimental design. The study was divided into two stages to simulate the mixed
infection of anthracnose (occurring annually from July to September) and powdery mildew
(occurring annually from August to October) in the greenhouse.
Stage 1: Stage 1 was designed to establish the anthracnose pathogen C. spinaciae-
infected (C
+
) and uninfected (C
−
) plants for stage 2. In this step, common vetch was either
infected or uninfected with C. spinaciae (30 pots for each treatment). The incidence and
disease index (DI) of anthracnose were assessed to determine the successful colonization of
C. spinaciae on the leaves of common vetch plants.
Stage 2: A fully crossed two-factor experiment was designed, which included C.
spinaciae (two levels)
×
soil (two levels) = four treatments, each with 10 replicate pots. The
disease incidence and DI of powdery mildew and the physiological and biochemical indices
of common vetch were determined after the spontaneous occurrence of powdery mildew.
2.2. Growth Medium
The common vetch was cultivated in two soils, including field soil (FS) with common
vetch, taken from the trial field in Xiahe County, and sterilized field soil (SFS) to establish
growth media with resident field soil microorganisms and exclude resident field soil
microorganisms, respectively. The soil mixtures were prepared by mixing 25% soil with
75% sand, which was then passed through a 2 mm sieve. The SFS and sands were sterilized
using autoclaving twice at 121
◦
C for 1 h for 3 d and then dried in an oven at 110
◦
C for 36 h.
The pots measured 14 cm (bottom diameter) by 22 cm (height) by 17 cm (top diameter) and
contained 2.5 kg of soil.
2.3. Experiment Establishment, Management, and Harvest
Stage 1. The seeds of “Lanjian No. 2” were surface-sterilized for 10 min using 10%
sodium hypochlorite (NaOCl), and then washed three times with sterilized distilled water.
The treated seeds were placed in sterile Petri dishes that contained sterile filter paper at the
bottom and incubated at 25
◦
C for 48 h to germinate. A total of 12 germinated seeds were
Plants 2024,13, 52 4 of 14
planted in each pot and thinned to eight seedlings of a similar size 1 week after emergence
for further experiments.
The Colletotrichum spinaciae was grown on potato dextrose agar (PDA). The conidia of
the pathogen were harvested in sterilized water from 30-day-old cultures that had been
incubated at 25
◦
C. The pathogen was inoculated 30 d after planting (DAP) using a spore
suspension of C. spinaciae that contained approximately 1.0
×
10
6
conidia per mL. The
numbers of spores in the solution were determined using a direct microscopic counting
procedure known as a Petroff-Hausser counting chamber (XB-K-25, Shiya Science and
Technology Company, Guangzhou, China). There were 30 pots each of C. spinaciae-infected
(C
+
) and C. spinaciae-uninfected (C
−
) plants. A suspension (20 mL) was sprayed onto the
C
+
plant (per pot), and the same volume of sterilized water was used for the C
−
plants. All
the plants were covered with a black plastic bag for 48 h, so they would be moist [
37
]. The
incidence and DI of anthracnose were measured and counted three times at 35, 40, and
45 DAP to determine the success of inoculation.
The severity of anthracnose disease was classified into six different levels based on
the percentage area of diseased lesions on the individual leaves: Level 0, no symptoms;
Level 1,
≤
10%; Level 2, 10.1–25.0%; Level 3, 25.1–50.0%; Level 4, 50.1–75.0%; and Level 5,
75.1–100%. The following equation was used to calculate the DI:
DI =∑(n×l)
N×L×100
where l,L,n, and Nrepresent the level of severity, the highest severity level, the number of
leaves for each severity level, and the total number of leaves, respectively.
Stage 2. Powdery mildew naturally infected all the plants at 46 DAP. The incidence
and DI of powdery mildew were then measured three times at 46, 51, and 56 DAP. The
number of total plants per pot and those diseased with powdery mildew were counted to
calculate the incidence by dividing the number of diseased plants by the total number of
plants. Moreover, all the leaves were counted to assess the DI of powdery mildew. The
classification and methods of calculating disease severity were the same as those described
in stage 1 for anthracnose, we used the percentage area of powdery mildew instead of
diseased lesions on the individual leaves.
The photosynthetic indicators, including the net photosynthetic rate, transpiration rate,
stomatal conductance, and concentration of intercellular carbon dioxide, were determined
every 8 d from 40 DAP onward (40, 48, and 56 DAP). A GFS-3000 portable photosynthetic
measuring system (Heinz Walz GmbH, Effeltrich, Germany) was used to measure the
photosynthetic indicators.
The plants in each treatment were mixed and sampled randomly before harvest. There
were four replicates, and each had more than 2 g of fresh leaves. These fresh leaf samples
were used to measure the activities of superoxide dismutase (SOD), polyphenol oxidase
(PPO), peroxidase (POD), and catalase (CAT). Approximately 0.1 g of fresh leaf sample was
required for each enzyme assay as described by Li et al. [
38
]. Coomassie brilliant blue and
uranine fluorescence were used to observe the powdery mildew mycelia as described by
Wang et al. [39].
The aboveground parts of the plants in every pot were obtained at 60 DAP using
scissors close to the soil and separated into different envelopes, dried, and weighed. The
fresh weight of each pot was determined immediately, and the dry weight was determined
after drying to a constant weight.
2.4. Statistical Analysis
The data are presented as the mean
±
SE. SPSS 20.0 (IBM, Inc., Armonk, NY, USA) was
used to analyze all the data. A two-way analysis of variance (ANOVA) was used to assess
the treatment effects on the incidence, DI, photosynthetic indices, and biomass production
with 10 replicates, except for the defense enzyme activities, which had four replicates.
Independent-sample t-tests were used to determine significant differences between the C
+
Plants 2024,13, 52 5 of 14
and C
−
plants in all the parameters and defined at the 95% confidence level. A Spearman’s
correlation heatmap was plotted using Origin 2022 (OriginLab, Northampton, MA, USA).
3. Results
3.1. Plant Fresh Weight and Dry Weight
The fresh and dry weights did not differ significantly between the C
−
and C
+
plants
(p> 0.05) (Figure 1). The fresh weight was lower in the SFS than in the FS (relative decrease
of 18.50%) (p< 0.05) (Figure 1a). In contrast, the dry weight was not significantly different
across all the treatments (p> 0.05) (Figure 1b), which indicated that the soil microorganisms
had a positive effect on the absorption of water by common vetch.
Plants 2024, 13, x FOR PEER REVIEW 5 of 14
The aboveground parts of the plants in every pot were obtained at 60 DAP using
scissors close to the soil and separated into different envelopes, dried, and weighed. The
fresh weight of each pot was determined immediately, and the dry weight was
determined after drying to a constant weight.
2.4. Statistical Analysis
The data are presented as the mean ± SE. SPSS 20.0 (IBM, Inc., Armonk, NY, USA)
was used to analyze all the data. A two-way analysis of variance (ANOVA) was used to
assess the treatment effects on the incidence, DI, photosynthetic indices, and biomass
production with 10 replicates, except for the defense enzyme activities, which had four
replicates. Independent-sample t-tests were used to determine significant differences
between the C+ and C− plants in all the parameters and defined at the 95% confidence
level. A Spearman’s correlation heatmap was ploed using Origin 2022 (OriginLab,
Northampton, MA, USA).
3. Results
3.1. Plant Fresh Weight and Dry Weight
The fresh and dry weights did not differ significantly between the C− and C+ plants
(p > 0.05) (Figure 1). The fresh weight was lower in the SFS than in the FS (relative
decrease of 18.50%) (p < 0.05) (Figure 1a). In contrast, the dry weight was not significantly
different across all the treatments (p > 0.05) (Figure 1b), which indicated that the soil
microorganisms had a positive effect on the absorption of water by common vetch.
Figure 1. Fresh (a) and dry (b) weight of common vetch—uninfected (C−) vs. infected (C+) with
Colletotrichum spinaciae grown in field soil (FS) and sterilized field soil (SFS). Different lowercase
leers above the bars indicate a significant difference between the FS and SFS at the p < 0.05 level. ns
means there is no significant difference between FS and SFS.
3.2. Disease Incidence and Disease Index
The Colletotrichum spinaciae-uninfected plants (C−) did not have anthracnose
symptoms, while the C+ plants showed typical symptoms 5 d after inoculation with the
pathogen. The disease incidence of common vetch anthracnose increased daily and
reached 86.25~92.50% (Figure 2a). The DI showed a paern similar to that of the disease
incidence and reached 28.98~31.08 (Figure 2b). The DI and disease incidence did not
differ significantly among the different soil treatments (p > 0.05).
Figure 1. Fresh (a) and dry (b) weight of common vetch—uninfected (C
−
) vs. infected (C
+
) with
Colletotrichum spinaciae grown in field soil (FS) and sterilized field soil (SFS). Different lowercase
letters above the bars indicate a significant difference between the FS and SFS at the p< 0.05 level. ns
means there is no significant difference between FS and SFS.
3.2. Disease Incidence and Disease Index
The Colletotrichum spinaciae-uninfected plants (C
−
) did not have anthracnose symp-
toms, while the C
+
plants showed typical symptoms 5 d after inoculation with the pathogen.
The disease incidence of common vetch anthracnose increased daily and reached
86.25~92.50% (Figure 2a). The DI showed a pattern similar to that of the disease inci-
dence and reached 28.98~31.08 (Figure 2b). The DI and disease incidence did not differ
significantly among the different soil treatments (p> 0.05).
Plants 2024, 13, x FOR PEER REVIEW 6 of 14
Figure 2. Anthracnose incidence (a) and disease index (b) of common vetch grown in field soil (FS)
and sterilized field soil (SFS).
Powdery mildew occurred at 46 DAP. The incidence of powdery mildew increased
over time. Moreover, the incidence was significantly higher in the C− plants
(92.08~98.33%) than in the C+ plants (24.17%~55.83%) (p < 0.05) (Figure 3a). However, the
incidence of powdery mildew did not differ significantly among the different soils under
the same treatment (p > 0.05). The DI of powdery mildew was lower in the C+ plants
(1.81~8.09) than in the C− plants (12.85~31.84) (p < 0.05) (Figure 3b). The soil treatment had
no impact on the DI of powdery mildew in both the C+ and C− plants (p > 0.05).
✱
✱
✱
✱
✱✱
Figure 3. Powdery mildew incidence (a) and disease index (b) in common vetch uninfected (C−)
and infected (C+) with Colletotrichum spinaciae grown in field soil (FS) and sterilized field soil (SFS). *
significant difference between common vetch infected and uninfected with C. spinaciae at the p <
0.05 level on the same date.
3.3. Photosynthetic and Physiological Indices
The net photosynthetic rate was higher in the C− plants than in the C+ plants by
156.06%~359.30% at 40 DAP (before powdery mildew occurrence) (p < 0.05). However,
the net photosynthetic rate was lower in the C− plants than in the C+ plants by
6.93~18.01% at 56 DAP (after powdery mildew infection for 10 d) (p > 0.05). The effect of
powdery mildew alone was similar to the combined effect of anthracnose and powdery
mildew infections (Figure 4a).
Figure 2. Anthracnose incidence (a) and disease index (b) of common vetch grown in field soil (FS)
and sterilized field soil (SFS).
Plants 2024,13, 52 6 of 14
Powdery mildew occurred at 46 DAP. The incidence of powdery mildew increased
over time. Moreover, the incidence was significantly higher in the C
−
plants (92.08~98.33%)
than in the C
+
plants (24.17%~55.83%) (p< 0.05) (Figure 3a). However, the incidence of
powdery mildew did not differ significantly among the different soils under the same
treatment (p> 0.05). The DI of powdery mildew was lower in the C
+
plants (1.81~8.09)
than in the C
−
plants (12.85~31.84) (p< 0.05) (Figure 3b). The soil treatment had no impact
on the DI of powdery mildew in both the C+and C−plants (p> 0.05).
Plants 2024, 13, x FOR PEER REVIEW 6 of 14
Figure 2. Anthracnose incidence (a) and disease index (b) of common vetch grown in field soil (FS)
and sterilized field soil (SFS).
Powdery mildew occurred at 46 DAP. The incidence of powdery mildew increased
over time. Moreover, the incidence was significantly higher in the C− plants
(92.08~98.33%) than in the C+ plants (24.17%~55.83%) (p < 0.05) (Figure 3a). However, the
incidence of powdery mildew did not differ significantly among the different soils under
the same treatment (p > 0.05). The DI of powdery mildew was lower in the C+ plants
(1.81~8.09) than in the C− plants (12.85~31.84) (p < 0.05) (Figure 3b). The soil treatment had
no impact on the DI of powdery mildew in both the C+ and C− plants (p > 0.05).
✱
✱
✱
✱
✱✱
Figure 3. Powdery mildew incidence (a) and disease index (b) in common vetch uninfected (C−)
and infected (C+) with Colletotrichum spinaciae grown in field soil (FS) and sterilized field soil (SFS). *
significant difference between common vetch infected and uninfected with C. spinaciae at the p <
0.05 level on the same date.
3.3. Photosynthetic and Physiological Indices
The net photosynthetic rate was higher in the C− plants than in the C+ plants by
156.06%~359.30% at 40 DAP (before powdery mildew occurrence) (p < 0.05). However,
the net photosynthetic rate was lower in the C− plants than in the C+ plants by
6.93~18.01% at 56 DAP (after powdery mildew infection for 10 d) (p > 0.05). The effect of
powdery mildew alone was similar to the combined effect of anthracnose and powdery
mildew infections (Figure 4a).
Figure 3. Powdery mildew incidence (a) and disease index (b) in common vetch uninfected (C
−
)
and infected (C
+
) with Colletotrichum spinaciae grown in field soil (FS) and sterilized field soil (SFS).
*significant difference between common vetch infected and uninfected with C. spinaciae at the
p< 0.05
level on the same date.
3.3. Photosynthetic and Physiological Indices
The net photosynthetic rate was higher in the C
−
plants than in the C
+
plants by
156.06%~359.30% at 40 DAP (before powdery mildew occurrence) (p< 0.05). However, the
net photosynthetic rate was lower in the C
−
plants than in the C
+
plants by 6.93~18.01% at
56 DAP (after powdery mildew infection for 10 d) (p> 0.05). The effect of powdery mildew
alone was similar to the combined effect of anthracnose and powdery mildew infections
(Figure 4a).
Plants 2024, 13, x FOR PEER REVIEW 7 of 14
Anthracnose and powdery mildew significantly affected the plant transpiration rate.
For example, the transpiration rate was significantly lower in the C+ plants than in the C−
plants (relative difference: 87.18~293.43%) at 40 DAP (p < 0.05) (Figure 4b).
✱
✱
Figure 4. Net photosynthetic rate (a) and transpiration rate (b) in common vetch uninfected (C−)
and infected (C+) with Colletotrichum spinaciae grown in field soil (FS) and sterilized field soil (SFS). *
significant difference between common vetch infected and uninfected with C. spinaciae at the p <
0.05 level on the same date.
Anthracnose and powdery mildew significantly affected the plant stomatal
conductance. The stomatal conductance was significantly higher in the C− plants than in
the C+ plants by 90.63%~312.63% at 40 DAP (p < 0.05). The average stomatal conductance
was slightly higher in the C− plants than in the C+ plants by only 3.01% after powdery
mildew infection for 10 d (p > 0.05) (Figure 5a).
Anthracnose and powdery mildew did not significantly affect the intercellular
carbon dioxide (CO2) concentration of common vetch. The C− and C+ plants had different
intercellular CO2 concentrations on 40 DAP and 56 DAP (relative decrease; 12.49% and
12.94%, respectively). In contrast, the intercellular CO2 concentration did not differ
significantly across the treatments (p > 0.05) (Figure 5b).
✱
Figure 5. Stomatal conductance (a) and intercellular carbon dioxide concentration (b) in common
vetch infected and uninfected with Colletotrichum spinaciae grown in field soil (FS) and sterilized
field soil (SFS). * significant difference between common vetch infected and uninfected with C.
spinaciae at the p < 0.05 level on the same date. The soil treatment had no impact on the
photosynthetic and physiological indices of common vetch when exposed to anthracnose and
powdery mildew (p > 0.05).
Figure 4. Net photosynthetic rate (a) and transpiration rate (b) in common vetch uninfected (C
−
)
and infected (C
+
) with Colletotrichum spinaciae grown in field soil (FS) and sterilized field soil (SFS).
*significant difference between common vetch infected and uninfected with C. spinaciae at the
p< 0.05
level on the same date.
Plants 2024,13, 52 7 of 14
Anthracnose and powdery mildew significantly affected the plant transpiration rate.
For example, the transpiration rate was significantly lower in the C
+
plants than in the C
−
plants (relative difference: 87.18~293.43%) at 40 DAP (p< 0.05) (Figure 4b).
Anthracnose and powdery mildew significantly affected the plant stomatal conduc-
tance. The stomatal conductance was significantly higher in the C
−
plants than in the C
+
plants by 90.63%~312.63% at 40 DAP (p< 0.05). The average stomatal conductance was
slightly higher in the C
−
plants than in the C
+
plants by only 3.01% after powdery mildew
infection for 10 d (p> 0.05) (Figure 5a).
Plants 2024, 13, x FOR PEER REVIEW 7 of 14
Anthracnose and powdery mildew significantly affected the plant transpiration rate.
For example, the transpiration rate was significantly lower in the C+ plants than in the C−
plants (relative difference: 87.18~293.43%) at 40 DAP (p < 0.05) (Figure 4b).
✱
✱
Figure 4. Net photosynthetic rate (a) and transpiration rate (b) in common vetch uninfected (C−)
and infected (C+) with Colletotrichum spinaciae grown in field soil (FS) and sterilized field soil (SFS). *
significant difference between common vetch infected and uninfected with C. spinaciae at the p <
0.05 level on the same date.
Anthracnose and powdery mildew significantly affected the plant stomatal
conductance. The stomatal conductance was significantly higher in the C− plants than in
the C+ plants by 90.63%~312.63% at 40 DAP (p < 0.05). The average stomatal conductance
was slightly higher in the C− plants than in the C+ plants by only 3.01% after powdery
mildew infection for 10 d (p > 0.05) (Figure 5a).
Anthracnose and powdery mildew did not significantly affect the intercellular
carbon dioxide (CO2) concentration of common vetch. The C− and C+ plants had different
intercellular CO2 concentrations on 40 DAP and 56 DAP (relative decrease; 12.49% and
12.94%, respectively). In contrast, the intercellular CO2 concentration did not differ
significantly across the treatments (p > 0.05) (Figure 5b).
✱
Figure 5. Stomatal conductance (a) and intercellular carbon dioxide concentration (b) in common
vetch infected and uninfected with Colletotrichum spinaciae grown in field soil (FS) and sterilized
field soil (SFS). * significant difference between common vetch infected and uninfected with C.
spinaciae at the p < 0.05 level on the same date. The soil treatment had no impact on the
photosynthetic and physiological indices of common vetch when exposed to anthracnose and
powdery mildew (p > 0.05).
Figure 5. Stomatal conductance (a) and intercellular carbon dioxide concentration (b) in common
vetch infected and uninfected with Colletotrichum spinaciae grown in field soil (FS) and sterilized field
soil (SFS). *significant difference between common vetch infected and uninfected with C. spinaciae
at the p< 0.05 level on the same date. The soil treatment had no impact on the photosynthetic and
physiological indices of common vetch when exposed to anthracnose and powdery mildew (p> 0.05).
Anthracnose and powdery mildew did not significantly affect the intercellular carbon
dioxide (CO
2
) concentration of common vetch. The C
−
and C
+
plants had different inter-
cellular CO
2
concentrations on 40 DAP and 56 DAP (relative decrease; 12.49% and 12.94%,
respectively). In contrast, the intercellular CO
2
concentration did not differ significantly
across the treatments (p> 0.05) (Figure 5b).
3.4. Enzyme Activities Related to Plant Defense
The enzymes related to defense in the C
+
and C
−
plants were determined after
powdery mildew infection. The activities of SOD, PPO, and CAT were higher in the C
+
than in the C
−
plants (relative increase; 147.88–148.21%, 182.50–196.25%, and 98.81–264.91%,
respectively) (p< 0.05) (Figure 6a,b,d). The activity of POD increased significantly in the
C
+
plants compared with the C
−
plants in the FS (p< 0.05), while there was no significant
difference in the SFS (p> 0.05) (Figure 6c). The values of the activities of SOD, PPO, and
CAT were higher in the SFS than in the FS, which indicated that soil microorganisms had a
negative impact on the activities of the plant defense enzymes (p< 0.05).
Plants 2024,13, 52 8 of 14
Plants 2024, 13, x FOR PEER REVIEW 8 of 14
3.4. Enzyme Activities Related to Plant Defense
The enzymes related to defense in the C+ and C− plants were determined after
powdery mildew infection. The activities of SOD, PPO, and CAT were higher in the C+
than in the C− plants (relative increase; 147.88–148.21%, 182.50–196.25%, and 98.81–
264.91%, respectively) (p < 0.05) (Figure 6a,b,d). The activity of POD increased
significantly in the C+ plants compared with the C− plants in the FS (p < 0.05), while there
was no significant difference in the SFS (p > 0.05) (Figure 6c). The values of the activities of
SOD, PPO, and CAT were higher in the SFS than in the FS, which indicated that soil
microorganisms had a negative impact on the activities of the plant defense enzymes (p <
0.05).
Figure 6. Defense-related enzyme activities of SOD (a), PPO (b), POD (c), and CAT (d) in common
vetch plants uninfected (C−) vs infected (C+) with Colletotrichum spinaciae grown in field soil (FS) and
sterilized field soil (SFS). Different lowercase leer above the bars indicates a significant difference
between common vetch infected and uninfected with C. spinaciae grown in field soil (FS) and
sterilized field soil (SFS) at the p < 0.05 level. CAT, catalase; POD, peroxidase; PPO, polyphenol
oxidase; SOD, superoxide dismutase.
3.5. Correlation Analysis between the Level of Infection and the Photosynthetic Index, Biomass,
and Defense Enzymes
There was a significant positive correlation between the disease incidence and DI for
both anthracnose and powdery mildew (p < 0.05). There was no significant correlation
between the disease incidence and DI of anthracnose and powdery mildew (p > 0.05). The
disease incidence and DI of powdery mildew significantly positively correlated with the
dry weight (p < 0.05) and significantly negatively correlated with the activities of POD,
PPO, CAT, and SOD (p < 0.05). In addition, the disease incidence of powdery mildew
significantly positively correlated with the intercellular carbon dioxide concentration
(ICDC) (p < 0.05) (Figure 7).
Figure 6. Defense-related enzyme activities of SOD (a), PPO (b), POD (c), and CAT (d) in common
vetch plants uninfected (C
−
) vs infected (C
+
) with Colletotrichum spinaciae grown in field soil (FS) and
sterilized field soil (SFS). Different lowercase letter above the bars indicates a significant difference
between common vetch infected and uninfected with C. spinaciae grown in field soil (FS) and sterilized
field soil (SFS) at the p< 0.05 level. CAT, catalase; POD, peroxidase; PPO, polyphenol oxidase; SOD,
superoxide dismutase.
3.5. Correlation Analysis between the Level of Infection and the Photosynthetic Index, Biomass, and
Defense Enzymes
There was a significant positive correlation between the disease incidence and DI for
both anthracnose and powdery mildew (p < 0.05). There was no significant correlation
between the disease incidence and DI of anthracnose and powdery mildew (p > 0.05). The
disease incidence and DI of powdery mildew significantly positively correlated with the
dry weight (p < 0.05) and significantly negatively correlated with the activities of POD, PPO,
CAT, and SOD (p < 0.05). In addition, the disease incidence of powdery mildew significantly
positively correlated with the intercellular carbon dioxide concentration (ICDC) (p < 0.05)
(Figure 7).
Plants 2024,13, 52 9 of 14
Plants 2024, 13, x FOR PEER REVIEW 9 of 14
Figure 7. Heatmap based on the Spearman’s correlation matrix of the biomass of fresh weight (FW)
and dry weight (DW), and the activities of POD, PPO, CAT, and SOD at harvest. The profiles before
harvest include the photosynthetic index of net photosynthetic rate (NPR) and stomatal
conductance (SC), transpiration rate (TR) and intercellular carbon dioxide concentration (ICDC),
the anthracnose incidence (AI) and anthracnose disease index (ADI), and the powdery mildew
incidence (PMI) and powdery mildew disease index (PMDI). CAT, catalase; POD, peroxidase; PPO,
polyphenol oxidase; SOD, superoxide dismutase. * statistical difference the two variables that
correspond to the small grid at the level of 0.05 (0.01 < p ≤ 0.05). ** statistically significant difference
at the level of 0.01 (0.001 < p ≤ 0.01). *** extremely significant statistical difference at the level of
0.001 (0.0001 < p ≤ 0.001).
4. Discussion
The interactions between synergistic diseases, such as anthracnose and powdery
mildew, can lead to unexpected dynamics in plant microbial multi-pathogen systems. In
this study, the response of common vetch infected with C. spinacia to the natural
development of powdery mildew was analyzed using a greenhouse experiment. The
plants infected with C. spinacia were less likely to be infected with powdery mildew.
Moreover, the plants that were first infected with C. spinacia had a lower severity of
powdery mildew symptoms, with a higher activity of defense-related enzymes (SOD,
PPO, POD, and CAT) involved in induced systemic resistance by anthracnose. Our
research confirmed the pre-infection interactions of the anthracnose pathogen to be
“ecological interference,” which reduced the contact rate and development to secondary
infection by powdery mildew. In addition, the damage caused by powdery mildew
infection alone and that caused by the combined infection with anthracnose and
powdery mildew were not significantly different.
The photosynthetic indices were lower in the C+ plants than in the C− plants before
powdery mildew infection, which indicated that anthracnose decreases the
Figure 7. Heatmap based on the Spearman’s correlation matrix of the biomass of fresh weight (FW)
and dry weight (DW), and the activities of POD, PPO, CAT, and SOD at harvest. The profiles before
harvest include the photosynthetic index of net photosynthetic rate (NPR) and stomatal conductance
(SC), transpiration rate (TR) and intercellular carbon dioxide concentration (ICDC), the anthracnose
incidence (AI) and anthracnose disease index (ADI), and the powdery mildew incidence (PMI)
and powdery mildew disease index (PMDI). CAT, catalase; POD, peroxidase; PPO, polyphenol
oxidase; SOD, superoxide dismutase. * statistical difference the two variables that correspond to
the small grid at the level of 0.05 (0.01 < p
≤
0.05). ** statistically significant difference at the
level of 0.01 (
0.001 < p≤0.01
). *** extremely significant statistical difference at the level of 0.001
(0.0001 < p≤0.001).
4. Discussion
The interactions between synergistic diseases, such as anthracnose and powdery
mildew, can lead to unexpected dynamics in plant microbial multi-pathogen systems. In this
study, the response of common vetch infected with C. spinacia to the natural development
of powdery mildew was analyzed using a greenhouse experiment. The plants infected
with C. spinacia were less likely to be infected with powdery mildew. Moreover, the plants
that were first infected with C. spinacia had a lower severity of powdery mildew symptoms,
with a higher activity of defense-related enzymes (SOD, PPO, POD, and CAT) involved
in induced systemic resistance by anthracnose. Our research confirmed the pre-infection
interactions of the anthracnose pathogen to be “ecological interference,” which reduced
the contact rate and development to secondary infection by powdery mildew. In addition,
the damage caused by powdery mildew infection alone and that caused by the combined
infection with anthracnose and powdery mildew were not significantly different.
The photosynthetic indices were lower in the C
+
plants than in the C
−
plants before
powdery mildew infection, which indicated that anthracnose decreases the photosynthesis
of common vetch. Anthracnose may affect photosynthesis by reducing the contents of
chlorophyll a and b and inhibiting the PSII photochemical activity [
18
]. Castro et al. found
that the Colletotrichum sp. decreases light capture and the assimilation of CO
2
in the
mesophyll, thus decreasing the photosynthetic performance [
40
]. Additionally, lower
Plants 2024,13, 52 10 of 14
gaseous exchanges in anthracnose-infected plants could be owing to the reduction in leaf
pigments, thus decreasing photosynthesis [41].
The photosynthetic indices of the C
+
and C
−
plants were approached gradually after
powdery mildew infection, indicating that powdery mildew suppresses the photosynthesis
of common vetch. Watanabe et al. found that severe infection with powdery mildew
significantly reduced the photosynthetic activity of the sprouts [
42
]. Powdery mildew
reduces photosynthesis by reducing the green leaf area through its visual lesions and
influencing the gas exchange of the remaining green leaf tissues [
43
]. Herein, there were
interactions between powdery mildew and anthracnose in common vetch, which could
also be affected by multiple fungal pathogens. However, Lopes and Berger (2001) showed
no interaction between rust and anthracnose [
44
], which indicates the diversity of plant
responses to infection with multiple pathogens.
The disease incidence and DI of powdery mildew differed significantly between the C
+
and C
−
plants, which indicated a significant interaction between the two diseases mediated
by common vetch under greenhouse conditions. The C
+
plants infected with pathogens
were less susceptible to powdery mildew than the C
−
plants, which indicates that common
vetch is more tolerant to powdery mildew after C. spinaciae infection. Similarly, Harth et al.
found that the infection of powdery mildew decreased in Cucurbita pepo after inoculation
with Zucchini yellow mosaic virus [
30
]. However, the soil did not induce significant
variation in the development of powdery mildew. Moreover, the leaves of common vetch
that had been pre-infected with anthracnose had substantially fewer powdery mildew
mycelia, possibly owing to the necrotic reactions of the plant hypersensitive response.
Pre-infection with C. spinaciae causes the death of plant tissue, which reduces the sites of
pathogen infection and the absorption of carbon from plants [
45
], which was similar to the
microscopic observations (Figures S1 and S2).
Plant diseases can cause serious losses in yield. For example, anthracnose causes
serious losses in the yield of velvetleaf (Abutilon theophrasti) [
46
], Chinese goldthread (Coptis
chinensis) [
37
], alfalfa (Medicago sativa) [
47
], plum (Pyrus spp.) [
48
], qing qian liu (Cyclocarya
paliurus) [
49
], and other crops in China. Similarly, powdery mildew reduces the production
of Euonymus japonicas [
50
] and wheat [
51
] spiny bitter gourd (Momordica cochinchinensis) [
52
],
and Jerusalem artichoke (Helianthus tuberosus) [
53
]. Notably, powdery mildew caused by
Erysiphe pisi,Erysiphe trifolii, and Erysiphe baeumleri can cause 25–50% yield losses [
54
]. A
previous study found that infection with anthracnose can significantly decrease the growth
and biomass of common vetch [
37
]. Herein, the biomass of the aboveground fresh and
dry weights was not significantly different between the C
+
and C
−
plants after infection
with powdery mildew, which confirmed the strong interaction between anthracnose and
powdery mildew. The infection of powdery mildew on common vetch is reduced by
anthracnose infection. Generally, the leaf abrasions caused by inoculation can provide
an easier avenue for the fungus to invade the plants [
30
]. However, powdery mildew
ingress seemed to be greatly reduced after anthracnose had already caused damage to
common vetch in our study. The two diseases caused the same damage as anthracnose
alone, which suggested that anthracnose could use some mechanism to prevent powdery
mildew invasion. Our hypothesis was partially upheld.
Plant defense enzymes strongly respond to pathogens [
55
]. The damage to the plant
leaves caused by pathogens leads to the formation of reactive oxygen species (ROS) [
56
],
which play a key role in plant defense. The accumulation of ROS can damage the plant
tissue [
57
]. Plants produce antioxidative enzymes, such as SOD, CAT, POD, and PPO, to
maintain the homeostasis of ROS by scavenging redundant ROS [
58
]. In this study, the
activities of enzymes related to defense were significantly higher in the C
+
plants than in
the C
−
plants. Moreover, infection with anthracnose upregulated the defense genes and
produced several enzymes related to defense [37,59–62].
Anthracnose induced a strong defense response and increased the activities of enzymes
related to defense, thus inhibiting the growth of powdery mildew. This explains why
powdery mildew was more severe in the C−plants than in the C+plants. Agents derived
Plants 2024,13, 52 11 of 14
from plants induce resistance to powdery mildew by activating the biochemical defense
of the treated leaves, such as by increasing the activities of PPO and POD [
63
]. This
was further supported by an analysis based on the Spearman’s correlation matrix of the
activities of the plant defense enzymes and the severity of powdery mildew. Chitosan
and potassium reduced the severity of powdery mildew and increased the activities of
PPO, POD, chitinase, and
β
-1,3-glucanase in okra (Abelmoschus esculentus) plants [
64
].
This mechanism related to plant defense enzymes has been used to develop resistance to
plant powdery mildew. For example, an extract of the brown alga Ascophyllum nodosum
significantly regulated the activity of defense-related enzymes, such as CAT, POD, and
PPO, to induce the active defense of squash (Cucurbita moschata) [
65
], strawberry (Fragaria
×ananassa) [23], and cucumber (Cucumis sativus) against powdery mildew [66].
The defense response in the C
+
plants induced by anthracnose may also lead to
the production of some secondary metabolites related to defense, such as phenolics and
flavonoids [
26
]. These metabolites and enzymes related to defense can improve the re-
sistance to powdery mildew. Moreover, this has been confirmed at the physiological,
biochemical, and genetic levels. Moreover, the levels of expression of the genes related to
hormone signaling, POD, and cell wall degradation under powdery mildew infection are
higher in the resistant varieties than in the susceptible varieties [67].
The resident soil microbiota did not affect the occurrence of anthracnose and the sub-
sequent natural development of powdery mildew. Equally, our hypothesis that common
vetch grown with resident soil microorganisms would have less severe disease was not
supported. The plants grown in unsterilized soil had higher fresh weights and lower
activities of plant defense enzymes, which suggested that the soil microorganisms had a
positive effect on the absorption of water by plants and plant defense to diseases. Recent
research has pinpointed the concept that enhanced nutrient uptake, improved root archi-
tecture, and protection of the host against biotic and abiotic stress are key functions of the
soil microbiome [
68
]. Further research is required to clarify the microbial mechanism in
complex plant–fungal pathogen interactions.
In conclusion, this study showed that anthracnose infection can establish “ecological
interference”, thus reducing the contact rate and development of powdery mildew infection.
The defense response and increase in the activities of defense-related enzymes, includ-
ing SOD, PPO, POD, and CAT, induced by anthracnose infection can thus improve the
plant immune response to powdery mildew. The physiological and biochemical changes
confirmed the interactive effect between anthracnose and powdery mildew in common
vetch. The resident soil microbiota from the growth of common vetch in the field had
no significant impact on the severity of plant disease, but they increased the activities of
the enzymes related to plant defense and showed a potential function to improve plant
disease resistance.
Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/plants13010052/s1, Figure S1: Morphological structures of powdery
mildew observed by commassie brilliant blue staining. a: mycelium structure of C
−
plant leaf; b:
mycelium symptoms of C
+
plant leaf; Figure S2: Morphological structures of powdery mildew
observed by uranine fluorescence staining. a: mycelium structure of C
−
plant leaf; b: mycelium
symptoms of C+plant leaf.
Author Contributions: Writing of original draft: F.L. and R.Z.; investigation: F.L.; graph and statistics:
F.L., R.Z. and F.G.; funding acquisition and revising of manuscript: T.D. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by the China Modern Agriculture Research System (CARS-22
Green Manure).
Data Availability Statement: Data are contained within the article and Supplementary Materials.
Conflicts of Interest: The authors declare no conflicts of interest regarding the publication of this paper.
Plants 2024,13, 52 12 of 14
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