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Virulent Fusarium isolates with
diverse morphologies show
similar invasion and colonization
strategies in alfalfa
Jian Yang
1
†
, Jing Han
2
†
, Yuqing Jing
1
†
, Siyang Li
1
,BoLan
1
,
Qian Zhang
3
and Kangquan Yin
1
*
1
School of Grassland Science, Beijing Forestry University, Beijing, China,
2
College of Forestry, Beijing
Forestry University, Beijing, China,
3
Lanzhou Institute of Husbandry and Pharmaceutical Science,
Chinese Academy of Agricultural Science, Lanzhou, China
Root rot is a major disease that causes decline of alfalfa production, and Fusarium
is a major pathogen associated with root rot. In this study, 13 Fusarium isolates
were obtained from alfalfa with root rot in Gansu Province, the major alfalfa
production region in China. The isolates were characterized by molecular
genotyping (ITS,TEF 1-aand RPB2 fragments) and identified as six species,
which included the F. acuminatum,F. incarnatum,F. oxysporum,F. proliferatum,
F. redolens, and F.solani. We found that their morphology varied significantly at
both the macro- and micro-levels, even for those from the same species. We
developed a low cost and fast pathogenicity test and revealed that all isolates
were pathogenic to alfalfa with typical root rot symptoms such as leaf yellowing
and brown lesions on the root and stem. However, the virulence of the isolates
differed. We also found that the conidia of all isolates germinated as early as 24
hours post inoculation (hpi), while hyphae colonized the root extensively and
invaded the xylem vessel by 48 hpi. Together our results reveal that different
virulent Fusarium isolates use a similar invasion strategy in alfalfa. This natural
plant-fungus pathosystem is intriguing and warrants further examination,
particularly with regard to efforts aimed at mitigating the impact of multiple
similar vascular pathogens on infected alfalfa plants.
KEYWORDS
fungal disease, root rot, forage crops, virulence, conidia, colonization
Introduction
Alfalfa (Medicago sativa L.) is known as the “queen of forage”with high nutritional value,
rich in vitamins and protein, and can prevent soil erosion and fixnitrogenefficiently (Barnes
et al., 1988;Veronesi et al., 2010;Li and Brummer, 2012). Therefore, alfalfa has a high feeding
value and economic benefits (Guo et al., 2019). To date, alfalfa has been planted more than 40
million hectares worldwide (Zhang et al., 2010). However, owing to large-scale cultivation,
Frontiers in Plant Science frontiersin.org01
OPEN ACCESS
EDITED BY
Giovanni Beccari,
University of Perugia, Italy
REVIEWED BY
Thabiso Eric Motaung,
University of Pretoria, South Africa
Jun Qin,
Northwest A&F University, China
Guoyong Xu,
Wuhan University, China
*CORRESPONDENCE
Kangquan Yin
yinkq@bjfu.edu.cn
†
These authors have contributed equally to
this work and share first authorship
RECEIVED 22 February 2024
ACCEPTED 30 April 2024
PUBLISHED 17 May 2024
CITATION
Yang J, Han J, Jing Y, Li S, Lan B, Zhang Q
and Yin K (2024) Virulent Fusarium isolates
with diverse morphologies show similar
invasion and colonization strategies in alfalfa.
Front. Plant Sci. 15:1390069.
doi: 10.3389/fpls.2024.1390069
COPYRIGHT
© 2024 Yang, Han, Jing, Li, Lan, Zhang and Yin.
This is an open-access article distributed under
the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that the
original publication in this journal is cited, in
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practice. No use, distribution or reproduction
is permitted which does not comply with
these terms.
TYPE Original Research
PUBLISHED 17 May 2024
DOI 10.3389/fpls.2024.1390069
alfalfa diseases have emerged (Wang et al., 2023). Among these, root
rot disease is one of the most devastating threats to alfalfa, contributing
to great production losses (Abbas et al., 2022). Root rot generally occurs
in alfalfa cultivation areas, particularly in North America, Argentina,
Russia,Australia,andJapan(Luo et al., 2019). In addition, root rot has
been reported in various regions of China, such as Gansu, Xinjiang, and
Heilongjiang provinces (Cai et al., 2021;Wang et al., 2021).
Root rot disease can be caused by various pathogens such as
fungi, nematodes, and bacteria (Cormack, 1937;Heydari et al.,
2012;Cao et al., 2020;Abbas et al., 2022). Fusarium spp. is one of
the most prevalent pathogens (Cormack, 1937). Fusaria are soil-
borne pathogens that can survive in soil and persist for a very long
time, causing damage to many agricultural crops (Noble and
Coventry, 2005). Furthermore, certain Fusarium species (for
example, F. culmorum and F. graminearum)canproduce
trichothecene mycotoxins (e. g. DON), which not only inhibit
germination, root growth, leaf mass of plants, seedling growth,
and regeneration, but also exert toxic effects on animals, including
feed refusal, induction of vomiting, growth retardation, increased
susceptibility to infections, reduced ovarian function, and
reproductive defects (Foremska et al., 1996;Rocha et al., 2005;
Habler and Rychlik, 2016;Inbaia et al., 2023). In the reproductive
history of Fusarium species, sexual and asexual spores act as
propagules that initiate infection (Ajmal et al., 2023). However,
far less than 20% of Fusarium species are sexually reproductive
(Ohara and Tsuge, 2004). The asexual spores are generally called
conidia, which are non-motile, walled, and haploid cells, and
include three types: macroconidia, microconidia, and
chlamydospores (Cole, 1986;Ohara and Tsuge, 2004).
Macroconidia are usually sickle-shaped and microconidia are
mostly oval or kidney-shaped. The chlamydospores were mostly
spherical and thick-walled. Not all Fusarium spp. produce all forms
of spores simultaneously (Ma et al., 2013). Conidia are critical in the
life cycle of Fusarium, as they can protect the genome under adverse
environmental conditions and serve as the primary means of
dispersion (Osherov and May, 2001). During the infection
process, the macroconidia and microconidia of Fusarium can
attach to the surface of plant rhizomes and spread to other plants
as secondary inocula and infectious agents (Rekah et al., 2000;
Ohara and Tsuge, 2004). Chlamydospores are more durable
survival structures in soil, more adaptable to adversity than
macroconidia and microconidia, therefore are more contagious
(Nicoli et al., 2013;Akhter et al., 2016). Taken together, conidia
play a crucial role in the occurrence and circulation of root rot, and
the quantity and growth rate of conidia also affect Fusarium
infection and colonization.
Upon infection, Fusarium can damage the alfalfa root tip,
leading to lesions of several colors: reddish, blackish, or brownish.
The root system could then gradually become soft and decayed,
accompanied by symptoms of severe chlorosis (Berg et al., 2017;
Wang et al., 2023). At the same time, alfalfa grows slowly and
eventually withers or even dies (Couture et al., 2002). And their
pathogenicity changes seasonally (Cormack, 1937). However, the
detailed infection strategy is unknown. Several methods have been
reported for evaluating the pathogenicity of Fusarium strains. One
of them is field evaluation, which mimics natural conditions
(Miller-Garvin and Viands, 1994). The second method is a soil
culture test conducted in a greenhouse or a growth chamber with
inoculated soil (Li, 2002;Infantino et al., 2006;Liu, 2006). The third
is the hydroponic screening method, which uses a liquid inoculum
suspension in specially designed pots with alfalfa seedlings under
growth chamber conditions (Cong et al., 2018). The current
pathogenicity assays are either time-consuming or affected by
microbial contamination, thus new methods are needed.
The aims of this study were (a) to identify pathogens associated
with alfalfa root rot combined with morphological and phylogenetic
analysis, (b) to establish a new method to evaluate the virulence of
different isolates, and (c) to reveal the detailed infection strategy of
Fusarium isolates in alfalfa.
Materials and methods
Plant materials and fungal isolates
Alfalfa plants with or without root rot symptoms were sampled
from Dunhuang, Gansu, which is the major alfalfa production
region in China (Figures 1A–C). The climate in this area is arid,
with sufficient sunshine, large temperature differences between day
and night (10–20°C), and low precipitation (40–200 mm/year).
Diseased roots were harvested and washed free of soil using tap
water. The roots were surface-sterilized with 70% ethanol for 10 s,
followed by 1% sodium hypochlorite solution for 3 min. After
surface sterilization, the roots were rinsed with sterile water three
times and then cut into 1 cm long segments with disposable knife
blades. To isolate fungi, we put four surface-sterilized alfalfa root
segments onto each 9 cm petri dishes on potato dextrose agar
(PDA) and incubated at 25°C in the dark for 5 days to allow fungal
growth. A total of four PDA plates were used. Then we picked the
emerging hyphae from the segments without bacteria
contamination and transferred them to new PDA plates to purify
single colony isolates.
DNA extraction, PCR amplification,
and sequencing
For DNA extraction, fungal cultures were grown on PDA for seven
days. A small amount of mycelia was carefully collected from the single
isolates using a sterile scalpel. Genomic DNA was extracted using the
CTAB method (Richards et al., 1994). The PCR amplification was
carried out for TEF 1-afragment with the primer EF1 (5’-
ATGGGTAAGGARGACAAGAC-3’)(O’Donnell et al., 2010)and
the primer EF2 (5’-GGARGTACCAGTSATCATG-3’)(O’Donnell
et al., 2010), for RPB2 fragment with the primer 5f2 (5’-
GGGGWGAYCAGAAGAAGGC -3’)(O’Donnell et al., 2010)and
the primer 7cr (5’-CCCATRGCTTGYTTRCCCAT -3’)(O’Donnell
et al., 2010)andforITS rDNA with the primer ITS1-F (5’-
Yang et al. 10.3389/fpls.2024.1390069
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CTTGGTCATTTAGAGGAAGTAA-3’)(Gardes and Bruns, 1993)
and the primer ITS4 (5’-TCCTCCGCTTATTGATATGC-3’)
(Vancov and Keen, 2009). The conditions for amplification of the
TEF 1-afragment were an initial denaturationstepof5minat95°C
followed by 35 cycles of denaturation (95°C for 30 s), annealing (48°C
for 30 s), and elongation (72°C for 30 s). The final elongation step was
carried out at 72°C for 5 min. The annealing temperatures of the RPB2
and ITS rDNA fragments were 52°C and 51.2°C, respectively. The
remaining amplification conditions were the same as those for the TEF
1-afragments. The PCR products were sequenced, and the obtained
sequences were blasted against the NCBI database.
Phylogenetic analysis
The nucleotide sequences obtained from Sanger sequencing
were aligned using ClustalW (Thompson et al., 2003) and manually
trimmed. The edited DNA Sequences of TEF 1-a,RPB2, and ITS
rDNA were concatenated for phylogenetic analysis. We used the
GTR + C + I default model of molecular evolution for maximum-
likelihood (ML) analyses and bootstrapping with 1000 replicates,
which were run using RAxML (Stamatakis, 2014). iTOL (ver3;
http://itol.embl.de/) was used to visualize the phylogenetic tree.
Virulence analysis
Alfalfa (cv. VISION) were used in virulence assays to evaluate
the virulence of Fusarium isolates using a fast “unimpaired root dip
inoculation on water agar”method at low cost. First, alfalfa seeds
were surface-sterilized with ethanol for 15 s, followed by three
washes with sterile water for 10 s. After removing the ethanol, the
seeds were surface-sterilized in a 10% household bleach solution
with 1% Triton-X for 15 min and washed three times with sterile
water. Surface-sterilized seeds were sown on water agar medium
(1% agar) and cultured in a growth chamber at 22°C under a 16 h
light/8 h dark cycle. To prepare the inoculation, fungal conidia were
collected from liquid potato dextrose cultures grown for five days by
passing them through two layers of Miracloth (Millipore,
Burlington, MA, USA). We measured conidia concentration
under a light microscope (10× objective) using a hemacytometer,
to which 10 mL of conidial solution was applied. A formula (conidia
concentration = N/5 × 25 × 10
4
per ml) was used to calculate
conidia concentration. N: the total number of conidia in the five
squares of the 25 grids zone (four corner squares and the middle
square) (Yang et al., 2023). And the conidial concentration was
adjusted to approximately 1 × 10
6
per ml. An equal volume (10 mL)
of fungal conidia was applied to each root tip of the alfalfa seedlings.
A
B
C
FIGURE 1
(A–C) Root rot symptoms in alfalfa grown in field. (A), healthy root. (B), brown lesions on the root and stem node of diseased alfalfa. (C), Black
streaking of the vascular system, showing necrotic symptoms. Bars = 1 cm.
Yang et al. 10.3389/fpls.2024.1390069
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Virulence of Fusarium was assessed by observing the degree of root
decay. After inoculation of Fusarium conidia, root of seedling of
alfalfa gradually developed lesions with different colors, including
brown, yellow or red. Some root crowns would soften and rot and
some plants showed chlorosis. We noticed that generally the degree
of root decay stabilized after 4 weeks of inoculation. Thus, virulence
was assessed by Diseased Grade four weeks after inoculation.
Disease Grade was calculated to assess the severity of plant
diseases and was classified on alfalfa roots as follows: Grade 1
“slight rotten”(0 to 25% area of root lesioned or rotted, the diseased
symptoms were the slightest, Grade 2 “rotten”(25% to 50% area of
root lesioned or rotted), Grade 3 “moderate rotten”(50% to 75%
area of root lesioned or rotted), and Grade 4 “severe rotten”(75% to
100% area of root lesioned or rotted, and seedling wilted or died)
(Xu et al., 2012;Wang et al., 2020). Virulence was assessed by visual
inspection of the severity of the lesions. 30 alfalfa seedlings were
inoculated in one experiment and repeated three times for
each isolate.
Histological observations
The Fusarium isolates were grown on PDA plates at 28–30°C
until sporulation. We collected mycelia of each Fusarium isolate
cultured on PDA medium, suspended them in ddH
2
O, and placed
them under a microscope to observe the conidia of each
Fusarium isolate.
Root sections inoculated for 24 h and 48 h were treated with
10% KOH for 45 min and neutralized with 2% HCL. After three
washes with 1 × PBS, root sections were placed in staining solution
(10 mg/ml WGA-fluorescein, 0.02% Tween20, 1 × PBS) overnight at
4°C and imaged using a Confocal Laser Scanning Microscope. And
we performed z-stack processing with CLSM at 48 hpi to
demonstrate that if hyphae inoculated the xylem. 3D visualization
movies were undertaken to analyze microscopy images using Imaris
software 9.2.0.
Results
Sample collection, fungi isolation and
molecular identification
While the vascular tissue of the healthy root showed a white
color (Figure 1A), the diseased root not only showed brown lesions
outside but also dark brown discoloration inside the vascular tissue
(Figures 1B,C). Diseased root tissues were plated on PDA plates for
fungal isolation. Thirteen fungal isolates belonging to six species
were identified from infected alfalfa roots based on three DNA
barcodes (Figures 2A–Z). Of these, 1C (Figures 2A,B), 2A
(Figures 2C,D), 2C (Figures 2E,F), 3A (Figures 2G,H) and 3B
(Figures 2I,J) were Fusarium proliferatum;1B(Figures 2K,L), 2D
(Figures 2M,N), 3C (Figures 2O,P) were Fusarium solani;1D
(Figures 2Q,R), 2B (Figures 2S,T) were Fusarium incarnatum;1A
(Figures 2U,V) was Fusarium acuminatum;2E(Figures 2W,X) was
Fusarium oxysporum;3D(Figures 2Y,Z) was Fusarium redolens.F.
proliferatum was the most frequently isolated species (38.5%),
followed by F. solani (23.1%) and F. incarnatum (15.4%).
Morphological characterization of
fungal isolates
The isolates on PDA were circular and formed cottony, and
aerial hyphae on the surface. Morphology varied among the
different isolates in terms of margin color, growth rate of hyphae,
and shape of macroconidia and microconidia (Figures 2–4;
Supplementary Figure 1).
F. proliferatum
The colony appeared cottony or floccose with abundant aerial
mycelia (Figures 2A–J). The surface was initially white, but
sometimes turned pale purple or orange with age and alternated
dark purple or orange concentric rings on both the upper and lower
surfaces, except 2C had yellow lower surface. The edges of the
colonies were white or yellow in color. In addition, abundant
sporulation was observed after 3 days of hyphal growth. Growth
rate of mycelium was 10.810 ± 0.845 mm/day for 1C, 13.169 ± 0.861
mm/day for 2A, 7.053 ± 0.831 mm/day for 2C, 12.804 ± 0.427 mm/
day for 3A, and 11.720 ± 0.403mm/day for 3B, respectively
(Supplementary Figure 1A). Macroconidia were slender, fusiform,
hyaline, with size ranging from 9.603–22.869 × 1.909–4.444 mm
(mean ± SD = 12.844 ± 3.120 × 2.828 ± 0.718 mm) for 1C; reniform,
slender, hyaline, blunt at both ends, with size ranging from 12.999–
31.465 × 2.154–4.209 mm (mean ± SD = 17.223 ± 3.672 × 3.063 ±
0.541 mm) for 2A; straight, falciform, 1 septate, with size ranging from
16.173–30.449 × 1.560–5.231 mm (mean ± SD = 21.630 ±
3.535×3.113 ± 0.809 mm) for 2C; fusiform, 2 septate, with size
ranging from 11.413–33.388×1.543–4.190 mm(mean±SD=
17.146 ± 4.550 × 2.701 ± 0.598 mm) for 3A; fusiform, hyaline,
blunt at both ends, with size ranging from 9.807–16.254 × 1.178–
3.553 mm (mean ± SD = 12.187 ± 1.727 × 1.878 ± 0.505 mm) for 3B
(Figures 3A,C,E,G,I,4A,C). Microconidia of 1C, 2A, 3A and 3B
were reniform, hyaline and no septate, whereas 2C were oval and
hyaline (Figures 3B,D,F,H,J). The size of microconidia were ranging
from 3.090–6.240 × 0.987–2.599 mm(mean±SD=4.588±0.943×
1.797 ± 0.379 mm) for 1C, 4.121–8.595 × 1.117–2.807 mm (mean ± SD
= 6.171 ± 1.216×1.989 ± 0.427 mm) for 2A, 3.647–6.273 × 1.192–
2.511 mm (mean ± SD = 5.028 ± 0.712×1.795 ± 0.279 mm) for 3A,
3.109–6.201 × 0.852–2.339 mm (mean ± SD = 4.422 ± 0.754×1.288 ±
0.317 mm) for 3B, and 3.343–5.673 × 1.344–3.832 mm(mean±SD=
4.208 ± 0.567×2.242 ± 0.545 mm) for 2C (Figures 4B,D).
F. solani
The colony appeared white to yellow with cottony mycelia
(Figures 2K–P). Three isolates all had white concentric rings on the
upper surfaces, but had different colors on the lower surfaces: 1B
had the brown concentric rings (Figures 2K,L), 2D had the milky
yellow concentric rings (Figures 2M,N) and 3C had the white
concentric rings (Figures 2O,P). For F. solani, sporulation occurred
after 3 days of mycelium growth. Growth rate of mycelium was
Yang et al. 10.3389/fpls.2024.1390069
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12.581 ± 0.380 mm/day for 1B, 11.019 ± 1.635 mm/day for 2D, and
12.381 ± 0.848 mm/day for 3C (Supplementary Figure 1B).
Macroconidia were slender, straight to reniform, hyaline, blunt at
both ends, with size ranging from 8.158–17.948 × 2.473–4.578 mm
(mean ± SD = 12.032 ± 2.410 × 3.215 ± 0.530 mm) for 1B; slender,
falciform, with tapering apexes and foot shaped bases, and size
ranging from 11.873–36.633 × 1.471- 5.172 mm (mean ± SD =
18.214 ± 5.287×3.159 ± 1.002 mm) for 2D; falciform, hyaline, with
size ranging from 10.378–23.590 × 2.646–5.362 mm (mean ± SD =
16.876 ± 3.950×3.465 ± 0.688 mm) for 3C (Figures 3K,M,O,4A,C).
Microconidia were straight to reniform, with size ranging from
4.437–9.277 × 1.610–3.768 mm (mean ± SD = 6.599 ± 1.402 × 2.684
± 0.609 mm) for 1B; ellipsoid, hyaline, with size ranging from 3.287–
7.473 × 1.556–3.061 mm (mean ± SD = 5.045 ± 0.998×2.179 ± 0.485
mm) for 2D; reniform, hyaline, with size ranging from 3.122–7.377
× 1.428–3.160 mm (mean ± SD = 5.163 ± 1.225 × 1.831 ± 0.388 mm)
for 3C (Figures 3L,N,P,4B,D).
F. incarnatum
Colony appearance was cottony or floccose with abundant
aerial mycelia and was white with brownish yellow concentric
rings on lower surface (Figures 2Q–T). The edge of mycelium
was creamy yellow. The sporulation was observed after aerial
mycelium growing 3 days. Growth rate of mycelium was 12.415 ±
A
BDE
FG IHJ
KLMN
C
O
PQR ST
UVWXY
Z
FIGURE 2
(A–Z), morphological characteristics of Fusarium isolates. Colony’s upper and lower surfaces of Fusarium isolates grown on PDA incubated at 25 °C
for two weeks. (A, B):(1C), (C, D):(2A), (E, F):(2C), (G, H):(3A), (I, J):(3B), F. proliferatum;(K, L):(1B), (M, N):(2D), (O, P):(3C), F. solani;(Q, R):(1D), (S, T):
(2B), F. incarnatum;(U, V):(1A), F. acuminatum;(W, X):(2E), F. oxysporum;(Y, Z):(3D), F. redolens. Bars = 1 cm.
Yang et al. 10.3389/fpls.2024.1390069
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1.382 mm/day for 1D and 9.909 ± 1.459 mm/day for 2B
(Supplementary Figure 1C). The macroconidia were typical
falciform shape, with tapering apexes and blunt bases, 2 septate,
with size ranging from 12.298–28.730 × 2.739–5.676 mm (mean ±
SD = 20.315 ± 3.913×4.028 ± 0.758 mm) for 1D; straight to
falciform, 3 septate, blunt at both ends, with size ranging from
12.276–26.843 × 2.379–4.623 mm (mean ± SD = 21.360 ± 4.032 ×
3.368 ± 0.626 mm) for 2B (Figures 3Q,S,4A–C). The microconidia
were ellipsoid, hyaline, no septate, with size ranging from 2.935–
6.216 × 1.775–3.824 mm (mean ± SD = 4.847 ± 0.719 × 2.581 ±
0.435 mm) for 1D; oval, hyaline, no septate, with size ranging from
2.820–6.687 × 1.851–3.549 mm (mean ± SD =4.733 ± 0.964 × 2.594
± 0.503 mm) for 2B (Figures 3R,T,4B,D).
F. acuminatum
The colonies appeared cottony with abundant aerial hyphae
(Figures 2U,V). The surface was pink to purple and the edge was
yellow. Abundant sporulation was observed after aerial mycelium
growth for 3 days. The growth rate of the mycelia was 8.325 ± 0.352
mm/day (Supplementary Figure 1D). Macroconidia were slender,
straight to sickle-shaped with curved apexes and inconspicuous
basal heels, 3- to 6- septate, with size ranging from 22.581–49.788 ×
2.879–5.814 mm (mean ± SD = 40.030 ± 5.892×4.199 ± 0.868 mm)
(Figures 3U,4A,C). Microconidia were oval or sickle-shaped with
1- to 2- septate, with tapering apexes and blunt bases, and with size
ranging from 3.638–6.252 × 0.743–3.320 mm (mean ± SD = 4.822 ±
0.895×1.187 ± 0.519 mm) (Figures 3V;4B,D).
AB D
EF G
I
H
JKL
MN
C
OP
QR S T
UV WX
Y
Z
FIGURE 3
Morphological characteristics of macroconidia and microconidia of Fusarium isolates. Bars = 10 mm. (A, B):(1C), (C, D):(2A), (E, F):(2C), (G, H):(3A), (I, J):
(3B), F. proliferatum;(K, L):(1B), (M, N):(2D), (O, P):(3C), F. solani;(Q, R):(1D), (S, T):(2B), F. incarnatum;(U , V):(1A), F. acuminatum;(W, X ):(2E), F.
oxysporum;(Y , Z):(3D), F. redolens.
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Fusarium oxysporum
The colony appeared cottony or floccose with abundant aerial
mycelia, which varied in color from white to pale violet in
concentric rings on both upper and lower surfaces (Figures 2W,
X). The edges of the colonies were milky. The sporulation was
observed after aerial mycelium growing 3 days. The growth rate of
the mycelia was 12.750 ± 0.293 mm/day (Supplementary
Figure 1E). Macroconidia were slender, straight to flat, 0- to 1-
septate, with tapering apexes and foot shaped bases, and 12.309–
20.978 × 1.724–4.040 mm (mean ± SD = 15.683 ± 2.468 × 2.854 ±
0.606 mm) (Figures 3W,4A,C). Microconidia were oval, ellipsoid or
reniform, hyaline, with size ranging from 4.459–7.832 × 1.879–
3.322 mm (mean ± SD = 5.425 ± 0.930×2.766 ± 0.334 mm)
(Figure 3X;Figures 4B,D).
Fusarium redolens
Colony appearance was woolly or felt-shaped with abundant
aerial mycelia. Mycelium were white to yellow with the passage of
time and the edge was yellow (Figures 2Y,Z). F. redolens had white
concentric rings on the upper surface, while showed yellow on the
lower surface. The sporulation was observed after aerial mycelium
growing 3 days. Growth rate of mycelium was 10.515 ± 0.691 mm/
day (Supplementary Figure 1F). Macroconidia were straight to
fusiform, with slightly blunt apexes and inconspicuous basal
heels, with size ranging from 12.460–38.110 × 1.575–5.758 mm
(mean ± SD = 16.585 ± 5.558×3.235 ± 0.980 mm) (Figures 3Y,4A,
C). Microconidia were oval to ellipsoid, reniform, 0- to 1- septate,
with size ranging from 1.917–9.062 × 1.096–3.922 mm (mean ± SD
= 3.540 ± 1.847 × 2.181 ± 0.668 mm) (Figures 3Z,4B,D).
Phylogenetic relationship among isolates
of Fusarium spp. from alfalfa
Forty-three Isolates from seven species complexes were chosen
for phylogenetic study (Supplementary Table 1). As series of studies
have demonstrated that multilocus DNA sequence other than single
gene fragment should be used for accurately identifying and placing
novel fusaria within a precise phylogenetic framework (O’Donnell
et al., 2010), we used combined sequences of ITS, TEF1-a, RPB2
genes to construct maximum-likelihood phylogenetic tree
(Supplementary Table 1;Figure 5). These fragments have high
resolution of Fusarium species (O’Donnell et al., 2010;Najafzadeh
et al., 2020). For each isolate, we sequenced 361 bp for ITS, 333 bp
for TEF1-a,and399bpforRPB2. The tree was rooted with
Fusarium penzigii from the FDSC. Phylogenetic evolutionary
relationships among the seven Fusarium species complexes were
resolved by ML bootstrapping. These seven species complexes
included a number of relevant Fusarium species: FFSC (n = 12),
FOSC (n = 6), FRSC (n = 4), FTSC (n = 6), FIESC (n = 7), FSSC (n =
7) and FDSC (n = 1). Phylogenetic analyses of combined sequences
of ITS,EF1-a,RPB2 genes separated the 43 isolates into two distinct
A
B
DC
FIGURE 4
The length of macroconidia (A) and microconidia (B) and the width of macroconidia (C) and microconidia (D) of different Fusarium isolates. n = 20.
(1A), F. acuminatum; (1B), (2D), (3C), F. solani; (1C), (2A), (2C), (3A), (3B), F. proliferatum; (1D), (2B), F. incarnatum; (2E), F. oxysporum; (3D), F. redolens.
Yang et al. 10.3389/fpls.2024.1390069
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clades and six lineages, which corresponding to six species
complexes. Clade A included FFSC, FOSC FRSC, FTSC, and
FIESC. Clade B only included FSSC. We found strong bootstrap
support for three nodes separating species complexes in the ML
analysis: 92% ML bootstrap for node separating FFSC and FOSC,
95% ML bootstrap for node separating FFSC, FOSC and FRSC, and
95% ML bootstrap for node separating FFSC, FOSC FRSC, FTSC,
and FIESC. In contrast, the FTSC received moderate bootstrap
support (60%) as a sister to the FIESC.
There were eight nodes with bootstrap support of 100%, whereas
23 nodes with bootstrap support below 60%. We found a full bootstrap
support for the node for separating three isolates (HQS38–9, MLFR-09,
and JFA12) and other nine isolates of FFSC. However, the bootstrap for
node separating these nine F.proliferatum isolates (SMFP3, 1C, 2A, 2C,
3A, 3B, E, H, and JZB3110233) was only 64%. Interestingly, all the five
isolates (1C, 2A, 2C, 3A, and 3B) found in diseased alfalfa root in this
study were less close to the above three isolates (HQS38–9, MLFR-09,
and JFA12). In the FOSC lineage, there was a sister relationship
between DHRL01 and other three strains (2E, JW277008,
JW277009) with a moderate bootstrap support (69%). In the FRSC
lineage, F. redolens isolate 3D clustered with 21SL97 and 22901
clustered with CBS743. In the FRSC lineage, 1A clustered with three
isolates of F. acuminatum (St551, S7–6, and S7–9) with 100% support.
In the FIESC lineage, F. incarnatum 1Dclusteredwith2Bwithstrong
bootstrap (99%). In the FSSC lineage, 2D was on the same branch with
P3 with 94% bootstrap support, while 1B and 3C clustered with 22820
and 123699 with very close relationship.
Virulence assay on seedlings under
sterilized conditions
To test whether each isolate was pathogenic to alfalfa, we
established an easy, low cost and fast method using only agar in
the recycled and autoclavable polypropylene boxes. To our surprise,
all isolates we obtained from the diseased alfalfa root were
pathogenic to alfalfa with typical root rot symptoms (Figure 6).
However, severity differed in isolates in term of degrees of leaf
yellowing, stunting, wilting and root brown lesion. Accordingly, we
calculated the incidence rate of grade 4 for each isolate and we
FIGURE 5
Phylogenetic tree of 13 Fusarium isolates from this study and 30 reference sequences from GenBank (Supplementary Table 1) based on the
concatenated partial sequences of the ITS,TEF 1-aand RPB2 genes. Letters indicate phylogenetic groups defined by 65% bootstrap support or
more. The phylogenetic tree was inferred using the maximum likelihood method based on General Time Reversible + C + I model.
Yang et al. 10.3389/fpls.2024.1390069
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defined virulence into three groups based on the following criteria:
if the incidence of grade 4 was above 25%, the isolate was grouped
into the highly virulent group; if incidence of grade 4 was between
10% and 25%, the isolate was grouped into the moderately virulent
group; if incidence of grade 4 was less than 10%, the isolate was
grouped into the slightly virulent group (Koyyappurath et al.,
2016)(Figure 6).
Isolate 1B, 2C and 2E were in the highly virulent group
(Figures 6A–C). Among them 2C caused the most severe root rot
symptom that root tips were blackened, while most of the rhizomes
appeared lesions with several colors: blackish or brownish, or even
turned reddish. In addition, all the root systems softened and rotted,
and all leaves were yellowed and moldy. The disease incidence at
grade 4 (severe rotten) of seedlings inoculation with 2C was more
than 50% (Figure 6O).
Most of the isolates were placed in the moderately virulent
group (Figures 6D–J), including 1A, 2B, 3C, 3D, and three isolates
of F. proliferatum (1C, 3A, 3B). We found that about half of the root
system showed brown lesions and leaves partially turned yellow.
1D, 2A and 2D were in the slightly virulent group (Figures 6K–
M). Among them, 1D was the most slightly virulent isolate that
causing far below 10% disease incidence at grade 4 (severe rotten) of
seedlings after inoculation (Figure 6O). In addition, only a small
portion of leaves of 1D showed yellowing symptoms, and the root
system showed slight discoloration (Figure 6K).
Proliferation and colonization of fungal
isolates on and in alfalfa roots
To investigate how Fusarium infect and colonize the alfalfa root,
for each isolate, we used FITC-WGA staining and CLSM to observe the
developmental processes associated with root infection. We found that
the conidia of 13 Fusarium isolates germinated as early as 24 hpi,
during which the bud tube emerged from the end of the conidia at the
surface of root cells (Figure 7). Among them, conidia of isolates 1A, 2A,
2D, and 3B grew into hyphae in a fast fashion, resulting in long and
slender hyphae (Supplementary Figure 2). Whereas the morphology of
AB D
EFG
I
H
JKL
MN
C
O
FIGURE 6
(A–N), symptoms of alfalfa seedlings after inoculation with Fusarium isolates for 4 weeks. N(CK), healthy plants without fungal inoculation. (A) (1B), F.
solani;(B) (2C), F. proliferatum;(C) (2E), F. oxysporum;(D) (1A), F. acuminatum; (E) (1C), G(3A), (H) (3B), F. proliferatum; F(2B), F. incarnatum;(I) (3C),
F. solani;(J) (3D), F. redolens; (K) (1D), F. incarnatum;(L) (2A), F. proliferatum;(M) (2D), F. solani.(O), Disease grades for infection assays of 13
Fusarium isolates at 4 weeks post inoculation. n = 30. Error bars indicated the SD of three biological replicates. Each small square has a side length
of 1 cm. Bars = 0.5 cm.
Yang et al. 10.3389/fpls.2024.1390069
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the germinated conidia of isolates 1B, 1C, 1D, 2B, 2C, 2E, 3A, 3C, and
3D was similar with tadpole shape, and their hyphae were shorter than
above four isolates (Supplementary Figure 2). As shown by arrows in
Figure 7, the shape of germinated conidia of isolates 1A, 1C, 2A, 2D,
2E, and 3A was flat and slender, and the shapes of other germinated
conidia of isolates 1B, 1D, 2B, 2C, 3A, 3C, and 3D were round or oval
(Figure 7).By48hpi,thehyphaewereirregularly distributed in the root
zone and successfully invaded the xylem (Figure 8). Among them,
more hyphae of isolates 1C, 2B, 2C, 2E, 3A, 3B, and 3C were distributed
onthexylem,andthesehyphaewerelongeranddenselyclustered
(Figure 8.1C,2B,2C,2E,3A,3B,3C).Moreover,thehyphaeofother
isolates (1A, 1B, 2A, 3D) were shorter and some of which distributed
on both sides of the xylem (Figure 8.1A,1B,2A,3D).Inaddition,
several germinated conidia and some shorter hyphae of isolate 1D were
observed at 48 hpi (Figure 8.1D).Comparedtootherisolates,the
number of hyphae in isolate 2D was the lowest (Figure 8. 2D).
Moreover, we found the hyphae of these isolates had reached the
xylem vessels through z-stack processing and 3D Visualization movies
(Figure 9;Supplementary Figures 3,4,Supplementary Movies 1–
Supplementary Movie 3).
Discussion
The primary soil-borne fungi that cause alfalfa root rot worldwide
are Fusarium, and thus most frequently isolated in rotted alfalfa root
(Leath et al., 1971;Jiang et al., 2021). Interestingly, however, the
composition of Fusarium species responsible for alfalfa root rot is
highly diverse in different regions, probably due to heterogeneous
environmental and geographical factors (Fang et al., 2019). In this
study, we isolated six Fusarium species (F. acuminatum,F. solani,F.
proliferatum,F. incarnatum,F. oxysporum and F. redolens)from
FIGURE 7
Confocal scanning laser microscopy images of root colonization by different Fusarium isolates on alfalfa roots at 24 hours post-inoculation. Arrows
indicate germinated conidia and asterisks indicate the germ tubes of Fusarium isolates. Bar = 25 mm. (1A), F. acuminatum; (1B), (2D), (3C), F. solani;
(1C), (2A), (2C), (3A), (3B), F. proliferatum; (1D), (2B), F. incarnatum; (2E), F. oxysporum; (3D), F. redolens.
Yang et al. 10.3389/fpls.2024.1390069
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diseasedrootsofalfalfainGansuprovinceofChina,whichisoneofthe
main production areas for alfalfa. Whereas in Inner Mongolia of China,
12 Fusarium species were identified with F. acuminatum most often
isolated (Wang et al., 2023). In addition, it was reported that F.
avenaceum,F. oxysporum and F. solani were most often isolated in
northeastern USA (Salter et al., 1994). As in Africa, it was reported F.
oxysporum,F. acuminatum, F. semitectum,F. fusariodes and F. equiseti
were isolated from roots in main production areas in Egypt (Seif El-
Nasar and Leath, 1983). These reports together with our current study
demonstrate that alfalfa root rot is a disease complex (Leath et al., 1971)
that can reduce yield in production areas, and also indicates the
difficulties in managing multiple pathogens in the same time for an
important perennial forage plant.
The morphological characters, including colony color, hyphae
growth rate, conidia morphology etc., are crucial for accurate
identification of Fusarium species (Summerell et al., 2003;
Jimenez-Fernandez et al., 2011). In this study, we found that each
isolate we isolated from alfalfa diseased roots both had
macroconidia and microconidia. However, the shape of their
macroconidia was very diverse ranging from the representative
sickle-shaped to straight, with length ranging from 8.158 mmto
49.788 mm. In contrast, the shape of their microconidia was mostly
oval without septate, with length ranging from 1.917 mm to 9.277
mm. Our result was similar to that of pathogen characterization of
post-flowering stalk rot in maize from agro-climatic zones of India,
in which 71 isolates from four Fusarium species were identified
(Harish et al., 2023). In addition, we found that the macroconidia
from F. acuminatum were the largest with length up to 49.788 mm
among our isolates. This was consistent with the findings of a
comprehensive investigation of global isolates of F. acuminatum,in
which the conidia produced by different isolates of F. acuminatum
were variable in length with a range of 38.2 mm-74mm for 5-
FIGURE 8
Confocal scanning laser microscopy images of root colonization by different Fusarium isolates on alfalfa roots at 48 hours post-inoculation. The
images reveal a dense mass of hyphae covering the root surface. Bar = 75 mm. (1A), F. acuminatum; (1B), (2D), (3C), F. solani; (1C), (2A), (2C), (3A),
(3B), F. proliferatum; (1D), (2B), F. incarnatum; (2E), F. oxysporum; (3D), F. redolens.
Yang et al. 10.3389/fpls.2024.1390069
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septate macroconidia, with some macroconidia up to 114.7 mm with
10 septa (Burgess et al., 1993). Although the aerial or plush mycelia
can reach the edge of the PDA dish (9cm) within two weeks, their
growth rate varied, even among isolates from the same species. The
slowest isolate was 2C (F. proliferatum) with hyphae growth rate at
7.053 ± 0.831 mm/day. Whereas the fastest isolate was 2A (F.
proliferatum) with hyphae growth rate at 13.169 ± 0.861 mm/day.
We speculate that these variabilities in daily mycelial growth could
be attributed to the presence of nutrient deficiencies (Robles-
Carrion et al., 2016). In addition, these isolates showed different
colors on PDA medium, even isolates from the same species showed
different colors, not only for the upper but also for the lower
surfaces of PDA plates. It is interesting to further explore the genetic
mechanisms of pigmentation differences among isolates from the
same species. The differences in conidia, hyphae growth rate and
pigmentation of isolates within species also press the need of
reliable molecular marker for accurate identification to species
level (O’Donnell et al., 2010).
Molecular marker plays the important role during the species
identification of Fusarium (O’Donnell et al., 2008). In this study, we
used three markers (ITS rDNA, TEF1-a,RPB2)toidentifyfungi
associated with alfalfa root rot disease. The combination of these
three markers could recognize all isolates from six clades (FFSC,
FOSC, FRSC, FTSC, FIESC, FSSC), but with varied bootstrap
support. For example, we found strong bootstrap support (100%) for
the FFSC, FTSC and FIESC clade, however the FOSC, FRSC and FSSC
clades received 86–92% bootstrap support. Moreover, it is difficult to
distinguish species withinthesameclade,suchasF. proliferatum and F.
annulatum in the FFSC clade, and F. solani and F. vanetteni in the
FSSC clade. To better recognize Fusarium species in the same clade,
combination of five gene datasets (CaM,rpb1,rpb2,tef1,andtub2)was
suggested to recognize all species within the FFSC (Yilmaz et al., 2021).
Whereas the combination of multi-locus dataset (ITS,TEF-1a,CAM,
RPB1,RPB2) was suggested to recognize all species within FIESC clade
(Wang et al., 2019). Recently, a total of 2020 strains isolated from
diseased cereal crops were successfully identified to 43 species using
multi-locus phylogeny, which including other new molecular barcodes
(CaM and H3)(Han et al., 2023). Thus, it is a great challenge to
discover universal barcodes for rapid and accurate identification of
Fusarium species (O’Donnell et al., 2010;Schoch et al., 2012;
Najafzadeh et al., 2020). As the pioneer research using 1001
homologous loci of 228 assembled genomes constructed a high-
confidence Fusarium species tree (Han et al., 2023), we envision that
multi-homologous loci based on whole genome sequencing will
provide robust classifications with higher resolution.
The development of a rapid and reliable laboratory technique to
assess the pathogenicity and virulence of numerous Fusarium strains
on alfalfa would be extremely beneficial. In this study, we used sterilized
FIGURE 9
Confocal scanning laser microscopy z-stack images showed hyphae penetrating xylem at 48 hours post-inoculation. Orthogonal views were
obtained from the areas indicated by white dotted line. The red square indicates that the hyphae reached the xylem. Bar = 200 mm. (1A), F.
acuminatum; (3C), F. solani.
Yang et al. 10.3389/fpls.2024.1390069
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water agar medium to evaluate the virulence of all the 13 Fusarium
isolates on alfalfa seedlings. Our method has several advantages. First,
sterilized condition excludes other microorganisms’effect on virulence,
which greatly improves the reproducibility of virulence assay. Second,
as water agar provide no or limited carbon source for most bacteria, it
can prevent growth of endophytic bacteria from alfalfa seeds, thus
greatly reduces bacteria contamination. Third, our method can test the
virulence in four weeks, which is faster than other methods with
months (Miller-Garvin and Viands, 1994;Infantino et al., 2006).
Therefore, our method has the potential to perform high-throughput
virulence assay for Fusarium on alfalfa, with the possibility extending to
other crop plants and even trees. In addition, this method can also be
used to test pathogenicity alteration of complex infection which
involves more than one isolates. Our research findings indicate that
while all 13 Fusarium isolates were capable of infecting alfalfa, their
virulence varied. It is worth noting that different isolates within the
same species exhibit varying degrees of virulence. For example, in the F.
proliferatum species, five isolates showed great virulent variations,
which is in agreement with previous reports on Fusarium pathogens
(Carter et al., 2002). We suspect that the variation in virulence among
isolates may be attributed to the differential expression of genes related
to virulence during the invasion of the isolates into alfalfa. Genetic
diversity has been observed among isolates (Taylor et al., 2016), and it
is possible that different isolates of the same species carry varying
virulent genes and exhibit different level of virulence when interacting
with host plants (Haapalainen et al., 2022). For instance, SIX1,thefirst
avirulence gene discovered in F. oxysporum,isnotpresentinall
isolates, indicating the association of effector genes profile with
virulence (Rep et al., 2004;Van Der Does et al., 2008;Lievens et al.,
2009). Therefore, comparative study in virulent genes (effector gene
identification, gene expression, etc.) among different isolates is
promising for unravelling the mechanisms of pathogenicity variation
in isolates.
The staining of 13 Fusarium isolates with a fluorescent dye
revealed that the conidia of these isolates germinated and produced
bud tubes within 24 hpi. Additionally, the hyphae of the isolates
covered the alfalfa roots, and the Fusarium species penetrated the
epidermal cells, further colonizing the xylem by 48 hpi. These
findings suggest that the penetration of the 13 Fusarium isolates
into the epidermis and xylem ducts of alfalfa roots occurred
between 24 and 48 hours. Similar infection processes have been
observed in other host plants, including conidial germination, bud
tube emergence, and hyphal invasion of the xylem ducts (Zvirin
et al., 2010;Guo et al., 2015). The nearly synchronized infection
process of the 13 different isolates on the same host plants suggests
that these isolates may have undergone concerted evolution in their
interactions with the host plants in terms of infection initiation and
colonization. This phenomenon also implies that different isolates
may cooperate to quickly overcome the host plant’s defense system.
Data availability statement
The datasets presented in this study can be found in online
repositories. The names of the repository/repositories and accession
number(s) can be found in the article/Supplementary Material.
Author contributions
JY: Investigation, Writing –original draft, Writing –review &
editing, Data curation, Validation, Formal Analysis, Visualization. JH:
Data curation, Investigation, Validation, Writing –review & editing.
YJ:Investigation,Writing–review & editing, Data curation, Software,
Validation, Visualization. BL: Data curation, Software, Writing –
review & editing. SL: Data curation, Investigation, Writing –review
& editing. QZ: Resources, Writing –review & editing. KY: Resources,
Writing –review & editing, Conceptualization, Funding acquisition,
Investigation, Project administration, Supervision, Writing –
original draft.
Funding
The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. This work
was supported by grants from the Fundamental Research Funds for
the Central Universities (2021ZY80) and Science and Technology
Innovation of Inner Mongolia Autonomous Region (2022JBGS0020).
Acknowledgments
The authors thank Prof. Yule Liu of Tsinghua University for
helpful suggestions and valuable discussions, Prof. Yonglin Wang of
Beijing Forestry University for guidance on fungal isolation, Prof.
Tiemei Wang of Beijing Forestry University for providing the alfalfa
seeds, Dr. Ling Chen of Beijing Forestry University for help in
collecting alfalfa plants, Dr. Yanbing Wang of University of
University of Georgia for editing our manuscript.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations,
or those of the publisher, the editors and the reviewers. Any product
that may be evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fpls.2024.1390069/
full#supplementary-material
Yang et al. 10.3389/fpls.2024.1390069
Frontiers in Plant Science frontiersin.org13
SUPPLEMENTARY FIGURE 1
The colony diameter of 13 isolates growing for different days. IBM SPSS
statistics 24 (https://www.ibm.com/support/pages/downloading-ibm-spss-
statistics-24) was used for st atistical analysis, includin g average co lony
diameters and corresponding standard deviations. Error bars indicated the
SD of three replicates. (A) (1C, 2A, 2C, 3A, 3B), F. proliferatum;(B) (1B, 2D, 3C),
F. solani;(C) (1D, 2B), F. incarnatum;(D) (1A), F. acuminatum;(E) (2E), F.
oxysporum;(F) (3D), F. redolens.
SUPPLEMENTARY FIGURE 2
The hyphae length of 13 isolates by 24 hpi. Error bars indicated the SD of at
least three replicates. 1D, 2B, 3A (n = 6); 2C (n = 5); 3B (n = 4); eight other
isolates (n = 3). (1A), F. acuminatum; (1B), (2D), (3C), F. solani; (1C), (2A), (2C),
(3A), (3B), F. proliferatum; (1D), (2B), F. incarnatum; (2E), F. oxysporum; (3D),
F. redolens.
SUPPLEMENTARY FIGURE 3
Confocal scanning laser microscopy z-stack images showed hyphae
penetrated xylem at 48 hours post inoculation. Orthogonal views were
made from areas indicated by white dotted line. The red square indicated
the hyphae had reached the xylem. Bar = 200 mm. (1D), F. incarnatum;
(3B), F.proliferatum.
SUPPLEMENTARY FIGURE 4
Confocal scanning laser microscopy z-stack images showed hyphae penetrated
xylem at 48 hours post inoculation. Orthogonal views were made from areas
indicated by white dotted line. The red square indicated the hyphae had reached
the xylem. Bar = 200 mm. (2E), F. oxysporum;(3D),F. redolens.
SUPPLEMENTARY TABLE 1
Fungal isolates and GenBank accession numbers used in the phylogenetic
analysis. ITS = internal transcribed spacer; TEF1-a= Elongation factor 1 alpha;
RPB2 = RNA polymerase IIsubunit.
SUPPLEMENTARY MOVIE 1
3D movie showed hyphae penetrated xylem at 48 hours post inoculation. Bar =
300 mm. 1D, Fusarium incarnatum.
SUPPLEMENTARY MOVIE 2
3D movie showed hyphae penetrated xylem at 48 hours post inoculation. Bar =
200 mm. 2A, Fusarium proliferatum.
SUPPLEMENTARY MOVIE 3
3D movie showed hyphae penetrated xylem at 48 hours post inoculation. Bar =
150 mm. 3C, Fusarium solani.
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