Introduction
Sphaeropsis sapinea Dyko & Sutton (syn. Diplodia sapinea
(Fr.) Fuckel, Diplodia
pinea (Desm.) Kickx.) causes shoot blight and canker diseases throughout
the world on conifers (Blodgett et al. 2005; Ye and He 2011). Disease
occurrence and its pathogenicity are economically important, affecting many
coniferous species, in particular Pinus spp. (Stanosz and Cummings 1996; Vornam
et al. 2019). According to CABI database, S. sapinea
is one of the most common fungal phytopathogen in
more than 65 countries in the world. S. sapinea
now is known to be widely distributed in the natural ranges of pines in the
Northern Hemisphere and where these trees have been introduced in the Southern
Hemisphere (Smith and Stanosz 2006). In China, it was
widely distributed in coniferous forest in thirteen provinces, which had caused
great economic and ecological losses (Ye and He 2011). The pathogen can invade
the host from wound and stomata of needles (Ye and He 2011). Through the
experiment of isolation and culture of fungi in tissue of Pinus
spp., Liu and Ye
(2003) found that the colonization of pathogen on the asymptomatic shoots in
winter may cause the incidence of shoot blight on spring shoots in the coming
year. Factors reducing the vigor of latently infected trees, for example, when
the host tree experiences environmental stress such as drought, have been shown
to trigger the latent infection becoming pathogenic, thereby causing severe tip
blight symptoms (Stanosz et al. 2001).
Endophytic fungi live in various tissues and organs of plants at a certain stage
or all stages of their life history, establishing harmonious associations with
plants. Some endophytes secrete antifungal and antibacterial metabolites at low
concentrations, thus inhibiting competitors (both endophytic
and pathogenic bacteria and fungi) and maintaining a balance of antagonism with
the competitors (Schulz et al. 2015; Suryanarayanan
et al. 2016). Multiple symbiosis between endophytic
fungi and host plants might result in reduced pathogen growth, as their growth
will be limited by secary metabolites of the endophytic fungi (Schulz et al. 2015). According to
Martinez et al. (2016), 36 genera of endophytic
fungi with antagonistic activity or metabolites have been isolated, but endophytic fungi that are difficult to cultivate or are unculturable still account for a high proportion. Most
studies concerning endophyte communities
have been based on pure cultures
isolated on artificial media, and it is difficult to determine the extent to
which their results are representative of natural infections in terms of
species abundance and occurrence.
In this study, whether S. sapinea can
affect the intact endophytic fungal community in
needles of P. densiflora was focused. The endophytic fungal diversity and community structure in
asymptomatic and symptomatic needles of P. densiflora
infected by S. sapinea were analyzed and
determined by high-throughput sequencing based on ITS (internal transcribed spacer) rRNA (ribosomal Ribonucleic Acid) gene. The community
structure and diversity of endophytic fungi were
analyzed, which provide a theoretical basis for the regulation of disease
microbial community structure. The followed assumptions were hypothesized: (i)-
endophytic fungal diversity in needles of P. densiflora differs with different levels of disease;
and (ii)- the community structure of endophytic fungi
is affected by S. sapinea infection.
Materials and
Methods
Study area and sampling
site
The Kunyushan Mountains are in the Jiaodong
Peninsula in Shandong Province in eastern
China (121°41'34"– 121°48'04" E; 37°11'50"–37°17'22" N). The Kunyushan Mountains are
the original habitat and natural distribution center of P. densiflora in China. In this region, P. densiflora and other coniferous species form a zonal
natural secary forest vegetation. Three standardized
plots of 30 m × 30 m plots were selected. The geographical coordinates of the
plots were 37°16'3.06" N, 121°45'32.60" E; 37o 15'57.94'N,
121°45'33.16" E; 37°15'52.17" N and 121°45'37.55" E.
The site conditions were all mid-slope, the slope is 23±3°, and the elevation was
360 m ± 30 m. Stand structure was a mixed coniferous forest, composed of P. densiflora and P. thunbergii.
Sample collection
Field sampling of
this study was carried out in three plots in September 2018, from which four
types of needle were collected: needles of asymptomatic P. densiflora (CZ1) and asymptomatic
needles of infected P. densiflora (CZ2), needles with a lighter level of
disease (where the length of the lesion was less than half of the length of the
needles; CZ3), and needles with heavier level of disease (where the length of
the lesion was more than half of the length of the needles; CZ4). The samples
were collected by a five-point sampling method (four vertices and the center
point of the plot), after which they were rinsed and dried. They were then
immersed in 75% ethanol for 1 min and rinsed with sterile water, soaked in 0.5%
sodium hypochlorite solution for 2 min, rinsed with sterile water, and stored
at -80°C.
Molecular detection of needle-associated fungal community
Sample DNA (deoxyriboNucleic acid) was extracted by the CTAB (cetyltrimethylammonium Ammonium Bromide) method, and
genomic DNA was extracted and detected by 1% agarose gel electrophoresis (Bullington and Larkin 2015). The DNA sample was thawed on
ice, centrifuged and thoroughly mixed. Then, a Nanodrop
ND-2000 (Thermo) was used to detect the sample quality, and 30 ng was taken for
PCR (polymerase chain reaction) amplification. The PCR amplification system was
as follows: DNA sample, 1 μL; forward primer (5 μM), 1 μL; reverse primer (5 μM), 1 μL; BSA
(Albumin from bovine serum) (2 ng μL-1), 3 μL; 2×Taq
Plus Master Mix, 12.5 μL;
ddH2O, 6.5 μL.
The amplification primer sequences of the ITS1 region were
(5'-CTTGGTCATTTAGAGGAAGTAA-3') and (5' -TGCGTTCTTCATCGATGC-3').
PCR was carried out using TransGen AP221-02
with TransStart Fastpfu DNA
polymerase. The PCR products of the same sample were mixed, detected by 2%
agarose gel electrophoresis, and cut with an AxyPrep
DNA Gel Recovery Kit (AXYGEN). The PCR product was recovered by gel, eluted
with Tris_HCl, and detected by 2% agarose
electrophoresis. Amplification products were constructed using the Illumina Miseq PE300 platform to
construct the Miseq library and to perform
sequencing.
Statistical analyses
of the fungal community
OTU (Operational
Taxonomic Unit) is artificially set to a certain classification unit (strain,
genus, species, group) in order to facilitate analysis in phylogenetic or
population genetics research. All sequences were clustered according to 97%
similarity with uclust (Version 1.2.22), and the OTU
of the singleton was removed, then the representative sequence and OTU table
were obtained (Youssef et al. 2009). OTU clustering was performed
according to 97% similarity sequence with usearch
(excluding single sequence), and the representative sequence was obtained. The
entire sequence was mapped out, according to 97% similarity, to form the OTU
list (Hess et al. 2011). Abundance and diversity index calculations of
OTU were performed using QIIME (v1.7.0) software to obtain species richness and
uniformity information within the sample. The PCA maps were drawn using R
(v2.15.3) software. All read sequences were deposited in the Sequence Read
Archive (SRA) of the National Coalition Building Institute (NCBI). The number
of data in SRA is SUB5584140.
Results
Fungal community
diversity and rarefaction curve
Following the
sequencing of needle samples for the entire fungal community, a total of 470375
high-quality sequences were obtained, which were clustered into 1045 OTUs in 12
samples. The OTU rarefaction curve of each sample tended to be smooth (see Fig.
1). The results of the coverage sequencing depth index of the sample are shown
in Table 1 (endophytic fungi). The results show that
the coverage for the collected needle samples was greater than 99.0%,
indicating that the fungal species information in the samples was fully tested
and that the test could represent the level of endophytic
fungi in the needles.
Analysis of community
structure of endophytic fungi in needles of P. densiflora
Based on the
number of OTUs, a Venn diagram (Fig. 2) and community structure histograms
(Fig. 3 and 4) were constructed to analyze the composition in four samples.
According to the Venn diagram, there were 429 identical OTUs in all samples,
accounting for 41.05% of the total number of OTUs.
The richness and Shannon diversity of endophytic
fungi were shown in Table 1. According to Chao1 index, CZ4 has the highest OTU
number, followed by CZ3, CZ1, and CZ2. As the disease worsened, the endophytic fungal richness showed an upward trend. The endophytic fungal diversity index of CZ1 and CZ2 was
similar, higher than CZ3, and the diversity index of CZ4 was the highest. The
results show that endophytic fungal abundance and
diversity increased with a longer lesion length.
Table 1: Abundance and diversity of endophytic fungi
in needles of P. densiflora
|
Chao1 |
Shannon |
Coverage |
|
|
CZ1 |
491.656 |
5.059 |
99.5% |
|
CZ2 |
491.068 |
5.059 |
99.5% |
|
CZ3 |
507.608 |
4.881 |
99.4% |
|
CZ4 |
549.363 |
5.231 |
99.5% |
Fig. 1: OTU-based rarefaction curve of endophytic
fungal communities in needles of P. densiflora
Fig. 2: Venn diagram of OTU distribution of endophytic
fungi in needles of P. densiflora
The 1045 OTU were classified into 29 phyla, 65classes, and 160 genera.
At the level of endophytic fungi in four types of conifers (CZ1, CZ2, CZ3 and CZ4), Ascomycota accounted for the highest proportion,
reaching 93.86, 89.95, 95.99 and 96.51%, respectively, followed by
Basidiomycota, which accounted for 3.69, 4.70, 1.86 and 1.85%, respectively.
The studied endophytic fungal communities were
largely dominated by Ascomycota.
At the class level (Fig. 3), endophytic fungi
in the four types of conifers (CZ1, CZ2, CZ3, and CZ4) were dominated by Dothideomycetes (39.63, 50.01, 35.97 and 28.90%,
respectively), where the sec relative abundance was that of Eurotiomycetes
(22.60, 11.83, 29.01 and 20.11%, respectively). Sordariomycetes,
Arthoniomycetes and Leotiomycetes
accounted for a relatively high abundance in the four samples, and the relative
abundance of Sordariomycetes (4.80, 4.67, 3.75 and
3.69%, respectively) and Arthoniomycetes (5.73, 5.84,
8.87 and 3.68%, respectively) were similar in different samples, while the
relative abundance of Leotiomycetes (5.42, 3.01, 3.98
and 30.57%, respectively) in CZ4 was significantly higher than the other three
samples. In addition to the above fungi, the other classes were different for
each sample. In CZ1 and CZ2, the proportion of Tremellomycetes
was 2.73 and 2.92%, respectively, while its relative abundance was less than
0.1% in CZ3 and CZ4. The relative abundances of Orbiliomycetes
and Taphrinomycetes were only higher than 1% in CZ2
and were lower than 1% in other samples.
At the genus level (Fig. 4), the dominant genera were different from
each other for every single sample. In CZ1, there were 12 genera with abundance
higher than 1%, the dominant genera were Paraconiothyrium
(9.03%), Selenophoma (7.39%), and Trichomerium (7.37%). In CZ2, there were 15
genera with abundance higher than 1%; the dominant genera were Sclerostagonospora
(13.08%), Paraphaeosphaeria (7.26%),
Phaeococcomyces (5.84%), and Selenophoma (5.25%). In CZ3, there were 9
genera with abundance higher than 1%; the dominant genera were Phaeococcomyces (8.87%), Trichomerium
(5.82%), Lapidomyces (5.07%), and Selenophoma (2.67%). In CZ4, there were 10
genera with abundance higher than 1%; the dominant genera were Cenangium (19.00%), Lophodermium
(10.32%), Trichomerium (5.85%), and Selenophoma (4.87%). Selenophoma, Trichomerium
and Phaeococcomyces occupied a certain
proportion in all samples. Cenangium and
Lophodermium were only
detected in CZ4.
Fig. 3: Dominant fungal classes from endophytic
fungi in needles of P. densiflora (the color of the column
represents the different classes, and the length of the column represents the
proportion size of the class. Sequences that could not be identified were
designated as “unidentified”. Genera making up less than 1% of total
composition in each sample were classified as “other”)
Beta diversity of endophytic fungi in needles of P. densiflora
Based on the
principal component analysis, the differences in endophytic
fungi from P. densiflora under different
infection conditions were evaluated (Fig. 5). The results show that the
contribution rates of principal component 1 (PC1) and principal component 2
(PC2) were 26.26 and 19.84%, respectively. Across the three plots, the distance
between the asymptomatic needles in the same plot was relatively close, and the
samples which were seriously infected by pathogens (CZ4) were concentrated,
indicating that the endophytic fungi in the needles
of P. densiflora differed between the plots,
but the infection of S. sapinea tended to make
the community structure of endophytic fungi
consistent.
Discussion
High-throughput
sequencing was used to analyze the diversity and community structure of endophytic fungi of P. densiflora
in mixed coniferous forests. The results show that the diversity of endophytic fungi in P. densiflora
needles was rich. In the present study, the dominant fungi were Ascomycota and
Basidiomycota. Ascomycota and Basidiomycota are very common and have been
reported as the dominant endophytic fungi of various
plant species (Deng et al. 2019; Yang et al. 2019).
Shoot blight of pine is one of the most common and widely distributed
diseases in conifers. Recent research on shoot blight of pine has included the
pathogens, transmission, infestation, prevention, and treatment (Lu 2017; An et al. 2018), as well as the diversity,
pathogenicity, and biological characteristics of the pathogens (Liu et al.
2018; Chen et al. 2019).
Fig. 4: Dominant fungal genera from endophytic fungi
of needles of P. densiflora (the color of the column
represents the different classes, and the length of the column represents the
proportion size of the class. Sequences that could not be u identified were
designated as “unidentified”. Genera making up less than 1% of total
composition in each sample were classified as “other”)
Fig. 5: Principal component analysis of endophytic
fungi in needles of P. densiflora
Control of the disease by
antagonistic bacteria has also progressed (Liu et al. 2012;
Wang and Ye 2016), and it has been confirmed that microbial regulation of host
plants can be achieved to prevent shoot blight of pine. Endophytic
fungal communities in asymptomatic tissues are more stable than that in
symptomatic tissues, which inhibits development of the pathogen. Zhang et
al. (2011) compared the differences of endophytic
fungal communities between asymptomatic and susceptible leaves in different
seasons through a culture-dependent method and analyzed the relationship
between gray spot disease and endophytic fungi
diversity in Camellia sinensis and the results
showed that the number, diversity, and evenness of endophytic
fungi in asymptomatic leaves were higher than those in infected leaves, and the
level of disease had significant effects on the diversity of endophytic fungi. Studies have also shown that Ascomycota
were found to be the most common fungal endophytes among all plant samples of
the two varieties (Rosa multiflora Thunb and R. multiflora var. carnea
Redouté and Thory), and the
Podosphaera pannosa (Wallr.) de Bary infection can
influence the fungal endophytic community, and some
of the endophytes may play a role in resistance (Zhao et al. 2018). The endophytic fungi of the lacquer-infected branch were
cultured, and the diversity of fungi in different parts was compared, and the
results showed that the endophytic fungal diversity
of asymptomatic branches was significantly higher than that of infected
branches (Takemoto et al. 2014). In the current study, the diversity of endophytic fungi showed first a trend of decreasing and
then increasing, and the highest diversity was observed in the heavily infected
samples. And there were differences in the diversity and community structure of
asymptomatic and infected needles. Endophytic fungi
diversity in asymptomatic needles was higher than in less infected needles,
which is consistent with previous research (Liu et al. 2016). When the
pathogen invades the tissue, the host plant's defense mechanism and its
internal endophyte balance are destroyed, therefore, other pathogenic fungi and
saprophytes are more likely to then enter the plant, which increases the
internal fungal diversity. In the study of endophytic
fungi in cotton roots infected with Verticillium
wilt, the endophytic fungi diversity of the roots
after infection with Verticillium wilt was
higher than that of healthy plants, indicating that the pathogens infection
increased the fungal diversity and affected its community structure (Liu et
al. 2016). This may be due to invasion of the diseased tissue by other
pathogens or saprophytic fungi (Arnold 2007) or as a result of the observed
pattern of the fungal colonies that can trigger the latent infestation of
pathogens (Steinrucken et al. 2016).
Most studies on the endophytic microbes of Pinus have been carried out by pure culture methods.
The endophytic fungi of P. sylvestris and P. koraiensis
have been isolated and cultured. The diversity of endophytic
fungi in pine needles and the factors affecting endophytic
fungal diversity, such as the age of coniferous leaves, were initially studied
(Dai and Lu 2012; Wang et al. 2017). In the study of endophytic
fungi in P. densiflora, multiple
dominant strains of Lophodermium complex, Sydowia polyspora, Hymenula spp., Sistotrema brinkmannii, Septoria pini-thunbergii, Earliella spp. and Lophodermium spp.
have been isolated (Gil et al. 2009; Eo et
al. 2013, 2018). Next generation sequencing technology has been frequently
used in the study of fungal diversity. To study the interaction between endophytic fungi in coniferous species, Bullington
and Larkin (2015) inoculated pathogens onto needles of P. monticola, and measured changes in endophytic
fungi diversity and community structure using new generation of high-throughput
sequencing methods. Interspecific competition and symbiotic patterns between
the inoculated fungi and the potential pathogens were also confirmed (Lu 2017).
The dominant species of endophytic fungi in
needles of P. densiflora in mixed coniferous
forest are Dothideomycetes and Eurotiomycetes.
At the genus level, Paraconiothyrium and Trichomerium are
common endophytic fungi found in Taxus
baccata (Somjaipeng et
al. 2015) and Coffea Arabica (Maharachchikumbura et al. 2018), and intensive
studies have been carried out on the metabolites produced by Paraconiothyrium spp., which showed moderate
antibacterial activity and restoration of the growth of a mutant yeast strain
inhibited by hyperactivated Ca2+ signaling
(Suzuki et al. 2019). Selenophoma spp. is a common pathogen in
ornamental plants (Sandoval et al. 2015) and crops (Kamlesh
and Kuldeep 2006), and its role in P. densiflora is still unclear. Cenangium
and Lophodermium were only found in the
infected needles, both of which have been found as pathogens in coniferous
species, such as P. sylvestris (Reignoux et al. 2014) and P. koraiensis (Ryu et al.
2018). Cenangium is a common genus of pine
disease and is a common pathogen in pine. This genus has been shown to be
closely related to S. sapinea infection (Milijašević and Karadžić
2004).
The fungal community may be affected by the specific composition of
tree species, which may contribute to alterations in the microenvironment
(Nguyen et al. 2016). Endophytic communities
of conifer species can vary, even between the needles of one tree (Deckert and Peterson 2000). Beta diversity analyses showed
that there was a difference among the asymptomatic needles of the three mixed
forest samples. After S. sapinea infection,
the community structure of endophytic fungi
tended to be consistent. For phytopathogenic fungi,
non-host tree species may act as barriers to spore transmission, resulting in a
‘dilution effect’ for fungal inoculum, thus, reduced fungal species richness
(Fernandez et al. 2019). For generalist fungi, several tree species may
act as alternative hosts, increasing the probability of successful
establishment and leading to differences between fungal community structure in
mixed forests.
Conclusion
Results showed
that endophytic fungal diversity and community
structure in P. densiflora needles were
affected by the S. sapinea infection in the
mixed coniferous forest. As the S. sapinea
infection worsened, the endophytic fungal richness
showed an upward trend. The dominant endophytic fungi
in P. densiflora needles from the mixed
coniferous forest are Dothideomycetes and Eurotiomycetes. Cenangium
was considered to have a certain association with the S. sapinea
infection. Future research should focus on the resistance of endophytic fungi to pathogens during infection.
Acknowledgments
This research was supported by the
National Key Research and Development Project of China (2018YFC1200400), the
CFERN & BEIJING TECHNO SOLUTIONS Award Funds on excellent academic
achievements, the operational grant of Kunyushan Forest Ecosystem Research
Station (2019132127) and the National Natural Science Foundation of China
(31270682). We thank Yingjun Zhang, Xiaowen Yuan, Bin Jiang, and Xin Song for
their help in collecting and handling the vast amount of data.
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