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205
Identication and fungicide sensitivity of Microdochium
chrysopogonis (Ascomycota, Amphisphaeriaceae), a new species
causing tar spot of Chrysopogon zizanioides in southern China
Xiang Lu1, Mengxian Mai1, Wenhui Tan1, Muyan Zhang1, Jie Xie1, Yi Lu1, Xue Li Niu1, Wu Zhang2
1 School of Life Sciences and Technology, Lingnan Normal University, Zhanjiang 524048, China
2 School of Geographical Science, Lingnan Normal University, Zhanjiang 524048, China
Corresponding author: Wu Zhang (ldzw1987@163.com)
Copyright: © Xiang Lu et al.
This is an open access article distributed under
terms of the Creative Commons Attribution
License (Attribution 4.0 International –
CC BY 4.0).
Research Article
Abstract
Vetiver grass (Chrysopogon zizanioides) has received extensive attention in recent years
due to its diverse applications in soil and water conservation, heavy metal remedia-
tion, as well as essential oil and phenolic acids extraction. In 2019, the emergence of
tar spot disease on C. zizanioides was documented in Zhanjiang, Guangdong Province,
China. Initially, the disease manifested as black ascomata embedded within leaf tissue,
either scattered or clustered on leaf surfaces. Subsequently, these ascomata became
surrounded by sheye lesions, characterised by brown, elliptical, necrotic haloes, which
eventually coalesced, resulting in leaf withering. Koch’s postulates demonstrated that
the fungus isolated from these lesions was the causal agent. Microscopic examination
showed that the pathogen morphologically belonged to Microdochium. The phyloge-
netic tree inferred from the combined ITS, LSU, tub2 and rpb2 sequences revealed the
three isolates including GDMCC 3.683, LNU-196 and LNU-197 to be a novel species of
Microdochium. Combining the results of phylogenetic, pathogenicity and morphological
analyses, we propose a new species named M. chrysopogonis as the causal agent of
C.zizanioides in southern China. The optimum growth temperature for M. chrysopogo-
nis was determined to be 30 °C. The in vitro fungicide sensitivity of M. chrysopogonis
was determined using a mycelial growth assay. Four demethylation-inhibiting (DMI) fun-
gicides, including difenoconazole, usilazole, propiconazole and tebuconazole and one
methyl benzimidazole carbamate (MBC) fungicide, carbendazim, were effective against
M. chrysopogonis, with mean 50% effective concentration (EC50) values of 0.077, 0.011,
0.004, 0.024 and 0.007 μg/ml, respectively. These ndings provide essential references
for the precise diagnosis and effective management of M. chrysopogonis.
Key words: fungicide sensitivity, multilocus phylogeny, new taxon, pathogenicity, tar spot
Introduction
Vetiver (Chrysopogon zizanioides) is one of the main grasses in tropical and
subtropical areas (Maurya et al. 2023). With a large root system that penetrates
deep into the soil, C. zizanioides is strongly tolerant to adverse environments,
such as drought, salinity and heavy metals. Recently, vetiver has been widely
Academic editor: C. Phukhamsakda
Received:
7 September 2023
Accepted:
19 November 2023
Published:
6 December 2023
Citation: Lu X, Mai M, Tan W, Zhang
M, Xie J, Lu Y, Niu XL, Zhang W (2023)
Identication and fungicide sensitivity
of Microdochium chrysopogonis
(Ascomycota, Amphisphaeriaceae),
a new species causing tar spot
of Chrysopogon zizanioides in
southern China. MycoKeys 100:
205–232. https://doi.org/10.3897/
mycokeys.100.112128
MycoKeys 100: 205–232 (2023)
DOI: 10.3897/mycokeys.100.112128
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MycoKeys 100: 205–232 (2023), DOI: 10.3897/mycokeys.100.112128
Xiang Lu et al.: New taxa of Microdochium from China
utilised in various applications, such as land restoration, soil and water con-
servation and phytoremediation of heavy metal-contaminated soils (Chen et
al. 2021). Moreover, the essential oil and phenolic acids extracted from vetiver
root possess signicant aromatic and biological properties, giving it an import-
ant role in perfumery, the food industry and medicine (David et al. 2019; Moon
et al. 2020).
To our knowledge, four diseases on C. zizanioides have been reported, name-
ly, leaf blight caused by Curvularia trifolii in India (Babu et al. 2021), root and
basal stem rot caused by Gaeumannomyces graminicola (Lin et al. 2019), leaf
spot caused by Phoma herbarum (Zhang et al. 2017) and leaf streak caused by
Stenocarpella chrysopogonis (Jia et al. 2023).
Microdochium species were originally introduced with the type species,
M.phragmitis, identied on the leaves of Phragmites australis in Germany (Sy-
dow 1924). Presently, there are 49 species included in this genus. However,
only a subset of these species can induce diseases, primarily affecting grass-
es and cereals. For example, M. albescens (also referred to as Monographella
albescens) typically induces leaf scald and grain discoloration in rice, leading
to a global reduction in rice yield (Araujo et al. 2016; Dirchwolf et al. 2023).
M. bolleyi is recognised for inducing root necrosis and basal rot in creeping
bent grass in Korea, as well as causing root rot on triticale in Kazakhstan (Hong
et al. 2008; Alkan et al. 2021). M. nivale and M. majus frequently result in the
occurrence of pink snow mould or Fusarium Patch on wheat, barley and turf
grass in cold to temperate regions (Ren et al. 2015; Abdelhalim et al. 2020).
M.opuntiae leads to brown spotting on Opuntia (Braun 1995). M. poae triggers
leaf blight disease in turf-grasses, such as Poa pratensis and Agrostis stolonif-
era (Liang et al. 2019). M. paspali is recognised for its ability to cause leaf blight
in seashore paspalum (Paspalum vaginatum) (Zhang et al. 2015). M. panatto-
nianum has the potential to induce anthracnose in lettuce (Galea et al. 1986).
M. sorghi is accountable for the formation of zonate leaf spots and decay on
sorghum species (Stewart et al. 2019).
The application of fungicides has always been an effective approach for dis-
ease control. In recent decades, demethylation-inhibiting (DMI) fungicides have
emerged as a signicant and extensive group of fungicides, exhibiting notable
ecacy in the control of diseases caused by the Microdochium genus. Notably,
compounds such as prochloraz, difenoconazole, propiconazole, metconazole,
myclobutanil, tebuconazole and triticonazole have shown substantial antifun-
gal ecacy against M. panattonianum, M. majus and M. nivale (Wicks et al.
1994; Debieu et al. 2000; Glynn et al. 2008; Mao et al. 2023). Additionally, fun-
gicide subgroups, including phenylpyrrole (PP) fungicides, such as udioxonil,
dicarboximides, such as iprodione and quinone outside inhibitors (QoIs), such
as trioxystrobin, have demonstrated noteworthy ecacy in the management
of diseases induced by M. nivale (Glynn et al. 2008; Koch et al. 2015; Aamlid et
al. 2017). Therefore, to promote effective control against tar spot of C. zizanioi-
des, it is necessary to determine the sensitivity of the pathogen to fungicides.
The main objectives of this study were to identify the pathogenic fungi caus-
ing tar spot of C. zizanioides in southern China on the basis of morphological
characteristics and multigene sequence analysis; to determine the pathogenic-
ity to C. zizanioides; and to determine the inhibitory effect of fungicides against
mycelial growth of the pathogen.
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Xiang Lu et al.: New taxa of Microdochium from China
Materials and methods
Sample collection and fungal isolation
Leaves exhibiting symptoms of tar spot on C. zizanioides were collected in elds
of the Grass Research Station of Lingnan Normal University (LNU), Zhanjiang,
Guangdong, China. Leaf segments (0.5 × 0.5 cm) from the transition zone from
diseased to healthy tissue were cut and surface-sterilised for 30 s with 75%
ethanol and 2% sodium hypochlorite (NaClO) for 1 min, rinsed with distilled wa-
ter 3 times, dried on sterile lter paper and placed on 2% potato dextrose agar
(PDA) (Crous et al. 2021b). Additionally, ascomata developing on the surface
of diseased tissue were gently scraped using a sterile scalpel. Subsequently,
a small number of ascospores were transferred and evenly spread on to the
surface of a water agar (WA) plate. Hyphal tips originating from leaf tissue
fragments and single germinating conidia were transferred on to PDA medium.
They were then incubated at 30 °C in darkness (Polizzi et al. 2009). After a
period of 7 days, the isolates were transferred on to PDA slants and preserved
at 4 °C in the culture collection of Lingnan Normal University. Additionally, they
were deposited in the Guangdong Microbial Culture Collection Center (GDMCC)
in Guangzhou, China. The holotype specimen was preserved in the Herbarium
of the Chinese Academy of Forestry (CAF) in Beijing, China.
Morphological characterisation
Colonies were subcultured on 2% malt extract agar (MEA) and oatmeal agar
(OA) at 30 °C for 10 days in the dark (Crous et al. 2009). Colony colour was
characterised using Rayner’s Mycological Color Chart (Rayner 1970) and colo-
ny diameters were measured after incubation for 10 days at 30 °C in the dark.
Morphological characters of ascomata, asci, ascospores, sporodochia, hyphae,
conidiomata, conidiophores, conidiogenous cells and conidia were determined
in sterile water using an Olympus BX53 compound microscope (Tokyo, Japan),
equipped with cellSens Dimension software (version 1.17).
DNA extraction, PCR amplication and sequencing
Fungal genomic DNA was extracted from mycelia grown on PDA medium after
10 days using the ENZA Fungal DNA Miniprep Kit (Omega Bio-tek, Doraville, Nor-
cross, GA, U.S.A.), according to the protocol of manufacturer. Four loci, including
internal transcribed spacer (ITS) rDNA region, large subunit ribosomal acid (LSU)
rDNA region, RNA polymerase II second largest subunit gene (rpb2) and part of
the beta-tubulin gene (tub2), were amplied by the following primer pairs: ITS1 and
ITS4 for ITS (White et al. 1990), LR0R and LR5 for LSU (Vilgalys and Hester 1990),
RPB150F (Jewell and Hsiang 2013) and fRPB2-7Cr (Liu et al. 1999) and Btub526F
and Btub1332R (Jewell and Hsiang 2013). The polymerase chain reaction (PCR)
conditions were as follows: 94 °C for 5 min; 94 °C for 30 s, annealing temperature
for 45 s and 72 °C for 1 min, 35 cycles; and a nal extension step at 72 °C for 10
min. The annealing temperature for ITS and LSU was 54 °C, for tub2 was 55 °C
and for rpb2 was 57 °C. PCR products were sequenced by Sangon Biotech Co.,
Ltd. (Shanghai, China). Sequences were edited and assembled using DNAMAN
version 5.2.2 and deposited in the NCBI GenBank nucleotide database (Table 1).
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Xiang Lu et al.: New taxa of Microdochium from China
Table 1. Strains included in the phylogenetic analyses with collection details and GenBank accession numbers.
Species Voucher Country GenBank Accession Number
LSU ITS tub2 rpb2
Microdochium albescens CBS 290.79 Ivory Coast KP858950 KP859014 KP859078 KP859123
CBS 291.79 Ivory Coast KP858932 KP858996 KP859059 KP859105
CBS 243.83 Unknown country KP858930 KP858994 KP859057 KP859103
M. bolleyi CBS 172.63 Germany MH869857 MH858255 – –
CBS 540.92 Syria KP858946 KP859010 KP859073 KP859119
Kaz_Mb01 Kazakhstan – MW301448 – –
Kaz_Mb02 Kazakhstan – MW301449 – –
CBS 137.64 Netherlands MH870023 MH858394 – –
CPC 25994 Canada KP858954 KP859018 KP859074 KP859127
CBS 102891 Germany MH874405 – – –
CBS 618.72 Germany MH872294 MH860598 – –
M. chrysanthemoides CGMCC 3.17929TChina KU746736 KU746690 KU746781 –
CGMCC 3.17930 China KU746735 KU746689 KU746782 –
M. chuxiongense YFCC 8794TChina OK586160 OK586161 OK556901 OK584019
M. citrinidiscum CBS 109067TPeru KP858939 KP859003 KP859066 KP859112
M. colombiense CBS 624.94TColombia KP858935 KP858999 KP859062 KP859108
M. dawsoniorum BRIP 65649TAustralia ON394569 MK966337 – –
BRIP 67439 Australia OM333563 MN492650 –ON624208
M. sheri CBS 242.90TUK KP858951 KP859015 KP859079 KP859124
NFCCI 4083 India KY777594 KY777595 – –
C30 ITI Sri Lanka – MT875317 – –
M. graminearum CGMCC 3.23525TChina OP104016 OP103966 OP236029 OP236026
CGMCC 3.23524 China OP104015 OP103965 OP242835 OP236026
M. hainanense SAUCC210781TChina OM959323 OM956295 OM981146 OM981153
SAUCC210782 China OM959324 OM956296 OM981147 OM981154
M. indocalami SAUCC1016TChina MT199878 MT199884 MT435653 MT510550
M. insulare BRIP 75114a Australia OQ892168 OQ917075 –OQ889560
M. lycopodinum CBS 146.68 The Netherlands KP858929 KP858993 KP859056 KP859102
CBS 109397 Germany KP858940 KP859004 KP859067 KP859113
CBS 109398 Germany KP858941 KP859005 KP859068 KP859114
CBS 109399 Germany KP858942 KP859006 KP859069 KP859115
CBS 125585TAustria KP858952 KP859016 KP859080 KP859125
M. maculosum COAD 3358TBrazil OK966953 OK966954 –OL310501
M. majus CBS 741.79 Germany KP858937 KP859001 KP859064 KP859110
10099 France – JX280597 JX280563 JX280560
10098 France – – JX280564 JX280561
99027 Canada – JX280583 JX280566 –
200107 Norway – KT736191 KT736253 KT736287
M. miscanthi SAUCC211092TChina OM957532 OM956214 OM981141 OM981148
SAUCC211093 China OM957533 OM956215 OM981142 OM981149
SAUCC211094 China OM957534 OM956216 OM981143 OM981150
M. musae CBS 143500TMalaysia MH107942 MH107895 MH108041 MH108003
CBS 143499 Malaysia MH107941 MH107894 MH108040 –
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Xiang Lu et al.: New taxa of Microdochium from China
Species Voucher Country GenBank Accession Number
LSU ITS tub2 rpb2
M. musae CBS 111018 Costa Rica – AY293061 – –
CPC 11240 Mauritius MH107944 MH107897 MH108043 –
CPC 16258 Mexico MH107945 MH107898 MH108044 –
CPC 11234 Mauritius MH107943 MH107896 MH108042 –
CPC 32681 Malaysia MH107946 MH107899 – –
M. neoqueenslandicum CBS445.95 The Netherlands KP858933 KP858997 KP859060 KP859106
CBS108926TNew Zealand KP858938 KP859002 KP859065 KP859111
M. nivale CBS 116205TUK KP858944 KP859008 KP859071 KP859117
200114 Norway – KT736185 –KT736279
200119 Norway – KT736199 KT736240 KT736263
200120 Norway – KT736210 KT736221 KT736273
200566 Norway – KT736220 KT736224 –
201050 Norway – KT736217 KT736236 KT736257
M. novae-zelandiae CBS 143847 New Zealand – LT990655 LT990608 LT990641
CPC 29693 New Zealand – LT990656 LT990609 LT990642
M. paspali CBS 138620TChina – KJ569509 KJ569514 –
CBS 138621 China – KJ569510 KJ569515 –
CBS 138622 China – KJ569511 KJ569516 –
M. phragmitis CBS 285.71TPoland KP858949 KP859013 KP859077 KP859122
CBS 423.78 Germany KP858948 KP859012 KP859076 KP859121
M. poae CGMCC3.19170TChina – MH740898 MH740914 MH740906
LC12115 China – MH740901 MH740917 MH740909
LC12116 China – MH740902 MH740918 MH740910
LC12117 China – MH740903 MH740919 MH740911
LC12118 China – MH740897 MH740913 MH740905
LC12119 China – MH740899 MH740915 MH740907
LC12120 China – MH740904 MH740920 MH740912
LC12121 China – MH740900 MH740916 MH740908
M. ratticaudae BRIP 68298TAustralia MW481666 MW481661 –MW626890
M. rhopalostylidis CBS 145125TNew Zealand MK442532 MK442592 MK442735 MK442667
M. salmonicolor NC14-294 South Korea MK836108 MK836110 – –
M. seminicola CBS 122706 Switzerland KP858943 KP859007 KP859070 KP859116
CBS 122707 Switzerland KP858947 KP859011 KP859081 KP859120
CBS 139951TSwitzerland KP858974 KP859038 KP859101 KP859147
KAS1516 Canada KP858961 KP859025 KP859088 KP859134
KAS3574 Switzerland KP858973 KP859037 KP859100 KP859146
KAS3158 Canada KP858970 KP859034 KP859097 KP859143
KAS1527 Canada KP858966 KP859030 KP859093 KP859139
KAS1473 Canada KP858955 KP859019 KP859082 KP859128
M. shilinense CGMCC 3.23531TChina OP104022 OP103972 OP242834 –
M. sinense SAUCC211097TChina OM959225 OM956289 OM981144 OM981151
SAUCC211098 China OM959226 OM956290 OM981145 OM981152
M. sorghi CBS 691.96 Cuba KP858936 KP859000 KP859063 KP859109
Microdochium sp. SAUCC1017 China MT199879 MT199885 MT435654 –
M. tainanense CBS 269.76TTaiwan KP858945 KP859009 KP859072 KP859118
CBS 270.76 Taiwan KP858931 KP858995 KP859058 KP859104
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Xiang Lu et al.: New taxa of Microdochium from China
Species Voucher Country GenBank Accession Number
LSU ITS tub2 rpb2
M. trichocladiopsis CBS 623.77TUnknown country KP858934 KP858998 KP859061 KP859107
M. triticicola RR 241 UK – AJ748691 – –
M. chrysopogonis GDMCC 3.683 China MT988024 MT988022 MW002441 MW002444
LNU-196 China MT988023 MT988020 MW002442 MW002445
LNU-197 China MT988025 MT988021 MW002443 MW002446
M. yunnanense SAUCC1018 China MT199880 MT199886 MT435655 –
SAUCC1015 China MT199877 MT199883 MT435652 MT510549
SAUCC1012 China MT199876 MT199882 –MT510548
SAUCC1011TChina MT199875 MT199881 MT435650 MT510547
Thamnomyces dendroidea CBS 123578 France KY610467 FN428831 KY624313 KY624232
Note: “T” denotes ex-type strain. Newly-generated sequences are indicated in bold. “-” means no data available in GenBank.
Phylogenetic analyses
The sequences of the strains from C. zizanioides and those of Microdochium spe-
cies, as well as the outgroup Idriella lunata obtained from NCBI GenBank, were
aligned with MAFFT version 7 using the default settings. Manual adjustments were
made to optimise the alignment in MEGA version 7.0 (Katoh and Standley 2013;
Kumar et al. 2016). To elucidate the taxonomic phylogenetic relationships, single
and concatenated ITS, LSU, rpb2 and tub2 sequence alignments were subjected to
analysis by applying Bayesian Inference (BI) using MrBayes version 3.2.5 and Max-
imum Likelihood (ML) using RAxML on the CIPRES portal (www.phylo.org) (Swof-
ford 2002; Crous et al. 2006; Ronquist et al. 2012). For BI analysis, the best evolu-
tionary model was determined through the utilisation of MrModelTest version 2.2
(Nylander 2004). Subsequently, in MrBayes v. 3.2.5, the Markov Chain Monte Carlo
180 (MCMC) algorithm was used to generate phylogenetic trees. The rst 25%
of saved trees were discarded as the burn-in phase. Posterior probabilities (PPs)
were determined from the remaining trees prior to calculation of the 50% majority
rule consensus trees; PP values exceeding 0.90 were considered signicant. The
ML analyses were performed by using RAxML-HPC BlackBox version 8.2.6, based
on 1000 bootstrap replicates. A general time reversible (GTR) model was applied
with gamma-distributed rate variation. Bootstrap values (BSs) equal to or higher
than 70% were regarded as signicant. The phylogenetic tree was viewed in Fig-
Tree version 1.4.4 (Rambaut 2018) and edited by Adobe Illustrator CC2018.
Pathogenicity test
Three isolates of M. chrysopogonis (GDMCC 3.683, LNU-196 and LNU-197)
were used to conduct the pathogenicity test. C. zizanioides plants were culti-
vated within a greenhouse, utilising plastic pots containing eld-collected soil
from the location where the plants had been established. The isolates were
cultured on PDA for 2 weeks at 30 °C in the dark to collect conidia.
For the detached leaf assay, 1-cm wide leaves were harvested from 2-month-old
plants cultivated in a greenhouse, washed under running tap water, surface disin-
fected with 70% ethanol for 1 min, rinsed with sterile water for 30 seconds and -
nally air-dried on sterilised lter paper. The conidial suspension was adjusted to a
concentration of 1 × 106 conidia/ml in sterile distilled water. An equivalent volume
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Xiang Lu et al.: New taxa of Microdochium from China
of sterile distilled water was used as a control. Leaf blades were then wounded
with a sterilised pin and each leaf was sprayed with 2 ml of conidial suspension.
All inoculated and control leaves were placed in a moist chamber at 25 °C with
100% relative humidity (RH) under cool uorescent light with a 12-h photoperiod.
After seven days, the disease incidence was assessed and calculated as the per-
centage of leaves with leaf tar spot symptoms. Each treatment consisted of ve
replicates and the experiment was conducted three times.
For the attached leaf assay, the leaf blades of healthy leaves were also pin-
pricked and the conidial suspension was adjusted to a concentration of 2 × 106
conidia/ml in sterile distilled water. An equivalent volume of sterile distilled wa-
ter was used as a control. In each treatment, ve plants were included, with
each plant being sprayed with approximately 20 ml of inoculum. All sprayed
and control plants were incubated in a plastic container in a greenhouse at 25 ±
2°C under cool uorescent light with a 12-h photoperiod. For the rst 3 days, the
plastic container was covered with transparent polyethylene bags to maintain
a high humidity. The disease incidence was assessed 10 days post inoculation
and calculated as the percentage of plants displaying tar spot symptoms. Each
treatment had three replicates and the pathogenicity test was repeated twice.
To full Koch’s postulates, symptomatic leaf tissues were subjected to sur-
face sterilisation as described above. Subsequently, these tissues were plated
on to PDA medium to enable the re-isolation of the fungi. These isolates were
identied, based on comparison of the cultures with those of the original strains.
Furthermore, the identications were conrmed by sequencing of the isolates.
Effect of temperature on mycelial growth rate
Mycelial growth rates of M. chrysopogonis isolates were assessed across vari-
ous temperatures. Mycelial plugs with a diameter of 5 mm were excised using
a sterile hole puncher from the periphery of 10-day-old PDA cultures. Subse-
quently, they were translocated to the central area of 90 mm PDA Petri dishes.
The cultures were subjected to incubation across a temperature range of 5, 10,
15, 20, 25, 30, 35, 40 and 45 °C. Four replicate plates per isolate were prepared
for each temperature. The plates were enveloped using Paralm (Bemis Com-
pany, Neenah, WI, U.S.A.) and then positioned within plastic containers prior to
their placement in incubators. The colony diameter was measured along two
mutually perpendicular axes and the mean of these two measurements was
documented as the radial colony diameter. Following a 10-day duration, myce-
lial growth rates were determined, based on colony diameter and subsequently
quantied in millimetres per day. Each treatment was replicated four times.
Fungicide sensitivity
To determine possible control measures for this pathogen in the eld, six groups
including nine fungicides were tested for their ability to inhibit the growth of M.
chrysopogonis in vitro. Fungicide sensitivity assays were conducted, based on
methods developed by Yin et al. (2019). The commercial formulations of fungi-
cides were serially diluted using sterilised distilled water. These diluted solutions
were added to autoclaved PDA medium that had been cooled to 55°C to obtain
the desired concentrations in micrograms per millilitre (Table 2). Isolates were cul-
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Xiang Lu et al.: New taxa of Microdochium from China
tured on PDA plates at 30 °C for 10 days in darkness to supply inoculum. Mycelial
discs (5 mm in diameter) from the periphery of colonies actively growing on PDA
were positioned at the centre of the fungicide-amended plates and unamended
(control) plates. The plates were then incubated at a temperature of 30 °C in dark-
ness for a duration of 2 weeks. Subsequently, the diameter of each colony was
measured along two perpendicular axes and the mean diameter was recalibrated
by deducting the diameter of the original plug utilised for inoculation. The effective
concentration for 50% mycelial growth inhibition (EC50) was estimated by perform-
ing a regression analysis of the percentage of mycelial growth inhibition against
the log10 of fungicide concentrations. Each treatment was replicated four times.
Statistical analysis
The dataset was tested for variance homogeneity using the Levene test. If
the variances were equal, an analysis of variance (ANOVA) followed by a least
signicant difference (LSD) test was conducted. In cases where the variances
were unequal, the Dunnett T3 test was applied. All statistical analyses were
carried out using IBM SPSS version 20.0 (SPSS Inc., Chicago, IL, U.S.A.). The
signicance threshold for detecting treatment disparities was set at P < 0.05.
Results
Disease symptoms and isolation of the pathogen
From 2019 to 2022, a previously unknown disease of vetiver grass occurred
during late spring and early autumn at the Grass Research Station of Lingnan
Normal University (LNU) in Guangdong Province, China. Symptoms consistent-
ly appeared on 85% of C. zizanioides grown under eld conditions. The initial
symptoms appeared as small and scattered punctate spots (< 1 cm) embed-
ded within the leaf tissue. Gradually, these spots clustered on leaf surfaces.
Subsequently, brown, elliptical, sh-eye necrotic haloes emerged, encircling the
lesion spots and aligning parallel to the leaf veins (Fig. 1A–C). As these ne-
crotic haloes coalesced, the leaf underwent chlorosis and wilting, eventually
leading to blighting of the entire plant. Ascomata were visible on the diseased
leaf surfaces.
Table 2. List of the fungicides used in this study.
Active ingredient Chemical family Trade name FRAC code Concentration (µg/ml)
Pyrimethanil anilino-pyrimidines Syngenta 9 0, 0.01, 0.1, 1, 10, 100
Difenoconazole triazoles Syngenta 3 0, 0.01, 0.1, 1, 10, 100
Fludioxonil phenylpyrroles Syngenta 12 0, 1, 10, 30, 100, 300
Iprodione dicarboximides Syngenta 2 0, 1, 10, 30, 100, 300
Flusilazole triazoles Syngenta 3 0, 0.001, 0.01, 0.1, 1, 10
Propiconazole triazoles BASF 3 0, 0.0016, 0.008, 0.04, 0.2, 1
Carbendazim benzimidazoles Syngenta 1 0, 0.0016, 0.008, 0.04, 0.2, 1
Metalaxyl acylalanines BASF 4 0, 10, 30, 100, 300, 1000
Tebuconazole triazoles Bayer 3 0, 0.0016, 0.008, 0.04, 0.2, 1
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Xiang Lu et al.: New taxa of Microdochium from China
Figure 1. Disease symptoms and morphological characters of Microdochium chrysopogonis on infected leaf tissue (CAF
800053) A–C tar spot symptoms of Chrysopogon zizanioides from natural infection in the eld D appearance of im-
mersed ascomata on infected leaves E ascomata in longitudinal section F–H asci I–N ascospores. Scale bars: 100 μm
(D, E); 10 μm (F–H).
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Xiang Lu et al.: New taxa of Microdochium from China
A total of 67 isolates were obtained on PDA. As the colony morphology of the
isolates was consistent, three representative isolates (GDMCC 3.683, LNU-196
and LNU-197), one from each eld, were selected for further studies.
Phylogeny
Based on a Megablast search on NCBI’s GenBank nucleotide database, the
closest hits for the ITS sequence of strain GDMCC 3.683 were M. dawsonio-
rum sequences with 98% identity (538/551, MK966337; 532/543, MN492650)
and a Microdochium sp. sequence with 97% identity (545/562, FJ536210).
The closest hits for LSU sequence of this strain were M. dawsoniorum se-
quences with 99% identity (868/871, OM333563; 864/867, ON394569) and a
M. yunnanense sequence with 99% identity (875/882, MT199880). The clos-
est hits for its rpb2 sequence were M. tainanense sequences with 85% identity
(711/841, KP859118 and KP859104) and a M. neoqueenslandicum sequence
with 83% identity (698/842, KP859106). The closest hits for tub2 sequence
were a M. tainanense sequence with 95% identity (661/697, KP859058), a
M.neoqueenslandicum sequence with 95% identity (665/703, KP859060) and
a M.colombiense sequence with 95% identity (658/695, KP859062). Therefore,
molecular analyses with all available Microdochium species were performed.
The alignment of each single locus and concatenated sequence dataset of ITS,
LSU, rpb2 and tub2 were used to conrm species resolution in Microdochium.
There were in total 99 aligned sequences, including the outgroup, Thamno-
myces dendroidea. A total of 3,033 characters (547 bp from the ITS, 843 bp from
LSU, 848 bp from tub2 and 795 bp from rpb2) were included in the phylogenetic
analyses. RAxML analysis of the combined dataset yielded a best scoring tree
with a nal ML optimisation likelihood value of -21, 329.537402 (ln). The matrix
had 1,096 distinct alignment patterns with 27.41% undetermined characters
or gaps. The tree length was 3.410120. Estimated base frequencies were: A =
0.234382, C = 0.267827, G = 0.258835, T = 0.238956; substitution rates were
AC = 1.101009, AG = 4.781387, AT = 1.240884, CG = 0.955029, CT = 6.933148
and GT = 1.000000; gamma distribution shape parameter α = 0.152657. Based
on the results of MrModelTest, the SYM + I + gamma for ITS, GTR + I + gamma
for LSU and rpb2 and HKY + I + gamma model for tub2 were selected as the
best t models for Bayesian analyses. A total of 47,402 trees were generated
by BI, amongst which 11,851 trees were discarded as the burn-in phase and
the remaining 35,551 trees were used to calculate the posterior probabilities
(PPs). The BI consensus tree conrmed the tree topology obtained with ML.
The well-supported clade (1/100) formed by the three strains from C. ziza-
nioides clustered with high support (1/100) with M. dawsoniorum (0.92/97),
which was sister to one single-strain clade representing M. ratticaude. This
clade clustered with high support (0.92/93) with the clade formed by M. albes-
cens, M. seminicola, M. graminearum, M. shilinense, M. insulare, M. paspali, M.
citrinidiscum, M.sorghi, M. tainanense and M. trichocladiopsis strains. The M.
neoqueenslandicum clade (1/100) was basal to this clade (Fig. 2). Single gene-
based phylogenies are presented in the Suppl. material 1. Nonetheless, these
individual gene trees did not yield a conclusive taxonomic classication for the
new species, in contrast to the comprehensive resolution achieved through the
concatenated sequence analysis.
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Xiang Lu et al.: New taxa of Microdochium from China
Figure 2. Phylogenetic tree inferred from a Maximum Likelihood analysis, based on a combined alignment of ITS, LSU,
tub2 and rpb2 sequences from 99 isolates of Microdochium sp. Bootstrap support values obtained with ML above 70%
and Bayesian (BI) posterior probability values above 0.90 are shown at the nodes (BI/ML). The tree was rooted to Tham-
nomyces dendroidea CBS 123578. Numbers of ex-type strains are emphasised with an asterisk and species are delimited
with shaded blocks. Isolates of M. chrysopogonis are indicated with lighter text.
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Taxonomy
Based on multilocus phylogenetic analyses, the three strains isolated from
C.zizanioides represent a previously unknown species within the genus Micro-
dochium that is closely related to M. dawsoniorum and M. ratticaudae. Morpho-
logical data placed the new species in the genus Microdochium. This species
is characterised below.
Microdochium chrysopogonis W. Zhang & X. Lu, sp. nov.
MycoBank No: 845624
Figs 1, 3
Etymology. Name refers to Chrysopogon, the host genus from which this fun-
gus was collected.
Description. Sexual morph on infected leaf tissue of the host plant (CAF
800054). Ascomata perithecial, 300–350 μm diam., solitary or in groups, im-
mersed, pale brown to black, subglobose to oval, uniloculate, non-ostiolate.
Paraphyses liform, hyaline, straight or curved, apically free. Asci 50–60 × 10–
18, x¯ = 55 × 13 μm (n = 50), hyaline, fasciculate, unitunicate, oblong to narrowly
clavate, fusiform, 8 biseriate spores with a short stipe. Ascospores clavate, hy-
aline, guttulate, 20–22 × 8–11.5, x¯ = 21 × 9 μm (n = 50), aseptate, smooth. Spo-
rodochia salmon-pink, slimy. Conidiophores reduced to conidiogenous cells.
Conidiogenous cells with percurrent proliferation, hyaline, smooth, aseptate,
ampulliform or obpyriform, 10–23 × 8–11.5, x¯ = 17 × 9.5 μm (n = 50). Conid-
ia fusiform, lunate, curved, solitary, guttulate, variable in length, 0–1-septate,
18–72 × 2–3.5, x¯ = 38.5 × 3 μm (n = 50), apex rounded, base usually attened.
Chlamydospores not observed. Vegetative hyphae on PDA (GDMCC 3.683) su-
percial and immersed, septate, branched, hyaline, smooth, 1–5.5 μm wide.
Culture characteristics. Colonies on PDA reaching 4.0–4.5 cm within seven
days in the dark at 30 °C, at, white cottony aerial mycelium, dense, saffron
rounded sporodochia produced after 3 weeks; reverse saffron. On MEA, sparse
white cottony aerial mycelium, orange rounded sporodochia produced; reverse
salmon-pink. On OA, periphery with white scarce cottony aerial mycelium, con-
centric rings of orange rounded sporodochia produced; reverse orange.
Type. China, Guangdong Province, Zhanjiang City, eld of the Grass Research
Station of Lingnan Normal University (LNU), from a leaf of vetiver grass (Chrys-
opogon zizanioides) with leaf tar spot disease, September 2019, W. Zhang & X.
Lu, holotype CAF 800054, ex-type living strain GDMCC 3.683.
Additional materials examined. China, Guangdong Province, Zhanjiang City,
eld of the Grass Research Station of Lingnan Normal University (LNU), from a
leaf of vetiver grass (C. zizanioides) with leaf tar spot disease, September 2019,
W. Zhang & X. Lu, strain LNU-196; China, Guangdong Province, Zhanjiang City,
eld of the Grass Research Station of Lingnan Normal University (LNU), from a
leaf of vetiver grass (C. zizanioides) with leaf tar spot disease, September 2019,
W. Zhang & X. Lu, strain LNU-197.
Notes. A multilocus phylogenetic analysis of the ITS, LSU, tub2 and rpb2 loci
placed three strains of M. chrysopogonis in a distinct and monophyletic clade
(1/100) sister to M. dawsoniorum and M. ratticaudae. Notably, M. chrysopogonis
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Figure 3. Microdochium chrysopogonis (from ex-type: GDMCC 3.683) A colonies after 7 days on PDA B colonies after 7
days on MEA C colonies after 7 days on OA D colony overview of the sporodochia on PDA in culture after incubation for
three weeks E aggregated conidiophores F conidiophores with conidiogenous cells G, H conidia. Scale bars: 20 μm (B).
has longer conidia (18–72 × 2–3.5 μm) than M. ratticaudae (7–11 × 1.5–2.5 μm)
and wider conidia than M. dawsoniorum (25–75 × 1–2 μm). Furthermore, the
conidia of M. chrysopogonis are guttulate and 0–1-septate, while those of M.
dawsoniorum are 0–3-septate and those of M. ratticaudae are aseptate. The
conidiogenous cells of M. chrysopogonis appear as percurrent, ampulliform or
obpyriform, whereas those of M. ratticaudae are indistinct from the hyphae and
those of M. dawsoniorum are cylindrical to irregular and exuous. Additionally,
the conidiogenous cells of M. chrysopogonis (10–23 × 8–11.5 μm) are wider
than those of M. ratticaudae (20–30 × 1–2 μm) (Table 3). Differences are also
evident in the sexual morph of these three species. In particular, the sexual
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morph is not observed in M. dawsoniorum. Ascomata size varies, with that of M.
ratticaudae (100–160 μm) being smaller than that of M. chrysopogonis (300–
350 μm). Ascospores of M. ratticaudae (14–24 × 4–7 μm) are fusoid to navic-
ular, while those of M. chrysopogonis are clavate, guttulate and wider (20–22 ×
8–11.5 μm). In addition, M. ratticaudae features abundant, pale to olivaceous
brown, subglobose or cylindrical chlamydospores, while these are not observed
in M. chrysopogonis (Crous et al. 2020, 2021a; Table 3). Consequently, based
on both morphological characteristics and phylogenetic analyses, all three iso-
lates of M. chrysopogonis were proposed as a new species.
Pathogenicity test
The symptoms observed on leaves of C. zizanioides after inoculation with the
representative isolate GDMCC 3.683 were similar to those observed in the eld.
No symptoms were observed on the leaves of the negative controls (Fig. 4).
The average disease incidence of detached leaves that were wounded and
sprayed with the isolates GDMCC 3.683, LNU-196 and LNU-197 was 93.3%,
80.0% and 93.3%, respectively. The average disease incidence of whole plants
after spraying with the same isolates was 76.7%, 73.3% and 73.3%, respec-
tively (Fig. 5). Koch’s postulates were fullled by successful re-isolation of the
fungal strains from all leaf spot tissues inoculated with the three isolates. The
morphology and DNA sequences of the isolates re-isolated from the inoculated
tissues were consistent with those of the strains used for inoculations.
Figure 4. Tar spot symptoms of Chrysopogon zizanioides 7 days after spraying on detached leaves (A, B) and 10 days
after spraying on leaves attached to whole plants (C, D) with Microdochium chrysopogonis isolate GDMCC 3.683 (B, D)
and sterilised water (A, C).
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Table 3. Morphological characters of Microdochium chrysopogonis and its related species.
Taxa M. albescens M. citrinidis-
cum
M. neoqueens-
landicum M. paspali M. semini-
cola
M. tricho-
cladiopsis
M. tainan-
ense M. sorghi M. dawsonio-
rum
M. ratticau-
dae
M. gramin-
earum
M. shilin-
ense
M. chrys-
opogonis
Asexual
morph
Conidia Shape falcate,
slightly to
strongly
curved, apex
pointed
cylindrical,
clavate,
obovoid
lunate,
allantoid,
curved
falcate, apex
pointed
cylindrical
to fusiform,
straight or
curved
oblong,
fusiform
to obovoid,
straight or
curved
lunate liform,
narrowly
acicular
fusiform,
obclavate
exuous
to falcate,
sometimes
with a
geniculation,
acute at the
tip, narrow at
the base
fusoid,
falcate,
acute at the
apex and
narrowed
at the base
n/a n/a fusiform,
lunate,
curved,
guttulate
Size
(μm)
11–16 ×
3.5–4.5
7–31 × 2–3 4–9 × 1.5–3 7–20.5 ×
2.5–4.5
19–54 ×
3–4.5
6–18 ×
2–3.5
10–15 ×
2–3
20–90 ×
1.5–4.5
25–75 × 1–2 7–11 ×
1.5–2.5
n/a n/a 18–72 ×
2–3.5
Septa 0–1(–3) 0–3 0(–1) 0–3 (0–)3(–5) 0(–1) 0–1 1–7(–10) 0–3 aseptate n/a n/a 0–1
Conidioge-
nous cells
Shape subcylindrical,
doliiform to
obpyriform
denticulate,
cylindrical
ampulliform,
lageniform to
subcylindrical
ampulliform,
lageniform to
cylindrical
ampulliform
to
lageniform
cylindrical
to clavate,
straight
but often
curved at
the tip
sympodial,
apical,
cylindrical or
ampulliform
with
conspicuous
rhachides
sympodial,
ovoid,
ampulliform
to obclavate
cylindrical
to irregular,
exuous,
narrowed
towards the
tip
indistinct
from
hyphae,
terminal,
solitary.
n/a n/a ampulliform
or
obpyriform
Size
(μm)
6–15 × 1.5–4 11–29 ×
1.5–2
4.5–10 × 2–3.5 6.5–15.5 ×
2.5–4
7–9.5 ×
3–4
4–37 × 2–3 3–10 × 1–3 5–13 × 3–4 20–30 × 1–2 n/a n/a 16.3–22.4
× 4.1–5.7
10–23 ×
8–11.5
Sexual
morph
Chla-
mydospores
Shape n/a n/a n/a n/a n/a present n/a n/a n/a subglobose
or
cylindrical
n/a n/a n/a
Perithecia Size
(μm)
150–180 ×
90–120
n/a n/a n/a 110–149 n/a n/a n/a n/a 100–160 n/a n/a 300–350
Asci Size
(μm)
40–85 ×
8–12
n/a n/a n/a 41–66 ×
7.6–11
n/a n/a n/a n/a 50–75 ×
10–14
55–77.5 ×
9.5–15.
50–76 ×
7–10
50–60 ×
10–18
Ascospores Size
(μm)
14–23 ×
3.5–4.5
n/a n/a n/a 12–22 ×
3–4.5
n/a n/a n/a n/a 14–24 ×
4–7
16.5–24 ×
4–5.5
14–18 ×
3–5.5
20–22 ×
8–11.5
Septa 1–3(–5) n/a n/a n/a 0–3 n/a n/a n/a n/a aseptate 0–3 0–3 aseptate
References Hernández-
Restrepo et
al. (2016)
Hernández-
Restrepo et
al. (2016)
Hernández-
Restrepo et al.
(2016)
Zhang et
al. (2015)
(Continued
on next page)
Hernández-
Restrepo et
al. (2016)
Hernández-
Restrepo et
al. (2016)
De Hoog &
Hermanides-
Nijhof (1977)
Braun
(1995)
Crous et al.
(2020)
(Continued
on next page)
Crous et al.
(2021a)
Gao et al.
(2022)
Gao et al.
(2022)
This study
Note: “n/a” means not provided in the literature.
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Figure 5. Disease incidence of tar spot symptoms on Chrysopogon zizanioides for leaves 7 days after spraying detached
leaves (A) and for whole plants 10 days after spraying leaves attached to potted plants (B), respectively, with Microdo-
chium chrysopogonis isolates GDMCC 3.683, LNU-196 and LNU-197. Values are shown as the means, with the error bars
representing the standard error. For each pathogen, columns with the same letter indicate means that are not signicant-
ly different according to a least signicant difference (LSD) test (P < 0.05).
Figure 6. Colony growth rate of three isolates, GDMCC 3.683, LNU-196 and LNU-197, of Microdochium chrysopogonis
from Chrysopogon zizanioides under different temperatures. Error bars represent the standard error.
Effect of temperature on mycelial growth
The mycelial growth of M. chrysopogonis was signicantly affected by tem-
perature (P < 0.01). All three isolates of M. chrysopogonis grew between 10 and
40 °C, with maximum growth observed at 30 °C (Fig. 6). No isolates grew at 5
or 45 °C after 3 days. The highest average mycelial growth rate was observed at
30 °C (26.5 ± 2.0 mm/day), followed by 25 °C (20.1 ± 4.7 mm/day).
Fungicide sensitivity
The EC50 values of various fungicides were analysed for their effectiveness
against M. chrysopogonis isolates. A total of 17 isolates of M. chrysopogonis
were collected from diseased leaves spanning the period from 2019 to 2022.
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The frequency distribution showed that difenoconazole, udioxonil, usi-
lazole, carbendazim and iprodione exhibited distributions resembling normal
curves, while pyrimethanil, propiconazole, metalaxyl and tebuconazole displayed
unimodal curves (Fig. 7). EC50 values for the inhibition of 17 M. chrysopogonis
isolates, based on mycelial radial growth, varied across fungicide treatments
(P < 0.05) (Table 4). Amongst the tested fungicides, usilazole had the lowest
EC50 values, with a notably concentrated response range of 0.001 to 0.007 µg/
ml and an average of 0.004 µg/ml. Tebuconazole closely followed with a slightly
narrower range, exhibiting values ranging from 0.002 to 0.009 µg/ml and an av-
erage of 0.007 µg/ml. Furthermore, there was no signicant difference between
usilazole and tebuconazole. Propiconazole displayed EC50 values spanning
from 0.006 to 0.016 µg/ml, with an average of 0.011 µg/ml, while those of car-
bendazim ranged from 0.008 to 0.031 µg/ml, with an average of 0.024 µg/ml. In
contrast, those of difenoconazole exhibited a broader range, varying from 0.013
to 0.127 µg/ml, with a mean value of 0.077 µg/ml, while those of pyrimethanil
ranged from 0.054 to 0.605 µg/ml, with an average of 0.411 µg/ml. Those of ip-
Figure 7. Frequency distribution of the 50% effective concentration (EC50) values of six groups including nine fungicides
for Microdochium chrysopogonis isolates, based on mycelial growth from 2019 to 2022.
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rodione, on the other hand, spanned from 15.018 to 260.335 µg/ml, with an aver-
age of 193.031 µg/ml. Metalaxyl exhibited the highest EC50 value, displaying the
widest range amongst all fungicides, extending from 302.785 to 1056.896 µg/
ml with an average of 892.677 µg/ml. Overall, these ndings indicate varying de-
grees of sensitivity to different fungicides amongst M. chrysopogonis isolates.
These variations in sensitivity could be essential considerations for designing
effective fungicide application strategies against vetiver leaf tar spot disease.
The inhibition of mycelial growth revealed that all nine fungicides exhibited
a reduction in fungal growth in vitro when compared to plates without amend-
ments. The effectiveness of these fungicides in diminishing the mycelial growth
of the isolates was contingent upon both the specic chemical compound and
its concentration. Four DMI fungicides, namely, difenoconazole, propiconazole,
usilazole and tebuconazole and one MBC fungicide, carbendazim, displayed
strong activity against M. chrysopogonis growth at concentrations below 1 µg/
ml, specically at concentrations of 1, 0.2, 0.1, 0.2 and 0.2 µg/ml, respectively
(Fig. 8). However, M. chrysopogonis showed a tendency to exhibit better growth
in the presence of pyrimethanil, udioxonil, iprodione and metalaxyl, with mycelial
growth being completely inhibited at concentrations exceeding 100 µg/ml (Fig. 8).
Discussion
In a survey of disease on C. zizanioides in Guangdong Province, China, from 2019
to 2022, tar spot was the predominant leaf spot disease. Isolation, morphological
features, multilocus phylogenetic analysis and pathogenicity tests conrmed that a
new Microdochium species, M. chrysopogonis was the causal agent. To effectively
control the disease, the sensitivity of M. chrysopogonis to six groups of fungicides,
including nine fungicides was determined. Results indicated that four DMI fungi-
cides, namely difenoconazole, propiconazole, usilazole and tebuconazole and
one MBC fungicide, carbendazim, were highly effective against the new species.
The morphology of the new species is introduced along with its sexual and
asexual morphological features, which are consistent with the following of
Microdochium: pale brown to black, subglobose to oval, uniloculate, perithecial
Table 4. In vitro sensitivity ranges and mean 50% effective concentration (EC50) values
for the inhibition of Microdochium chrysopogonis.
Fungicide EC50 (μg/ml)
Lowest Highest Mean ± SE
Difenoconazole 0.013 0.127 0.077 ± 0.039e
Pyrimethanil 0.054 0.605 0.411 ± 0.180d
Fludioxonil 0.014 6.128 4.525 ± 1.626c
Iprodione 15.018 260.335 193.031 ± 99.462b
Flusilazole 0.001 0.007 0.004 ± 0.003h
Propiconazole 0.006 0.016 0.011 ± 0.003g
Carbendazim 0.008 0.031 0.024 ± 0.009f
Metalaxyl 302.785 1056.896 892.677 ± 236.145a
Tebuconazole 0.002 0.009 0.007 ± 0.002h
Note: The letters indicate the comparison amongst the different fungicide treatments. Means followed by the
same letter do not differ according to a post hoc Dunnett T3 test (p < 0.05).
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Figure 8. Effect of fungicides on the mycelial growth of Microdochium chrysopogonis. Values are shown as the means,
with the error bars representing the standard error.
ascomata; hyaline, fasciculate, unitunicate, oblong to narrowly clavate, eight bise-
riate spores with short stipe asci, from which hyaline, clavate, smooth ascospores
arise. Conidiophores reduced to hyaline, smooth, aseptate, percurrent, ampulli-
form or obpyriform, conidiogenous cells, from which hyaline, 0–1-septate, fusi-
form, lunate conidia with the apex rounded and base attened usually arise (Figs
1, 3) (Hernández-Restrepo et al. 2016). The concatenated ITS, LSU, tub2 and rpb2
sequences were able to identify species in Microdochium and proved to be suit-
able barcoding markers in the process of species resolution (Hernández-Restrepo
et al. 2016). Phylogenetic analysis indicated that M. chrysopogonis formed a dis-
tinct well-supported clade (1/100) and was closely related to M. dawsoniorum and
M. ratticaudae (Fig. 2). Nevertheless, the classication of the new species in the
genus Microdochium is well supported by morphology, based on sexual and asex-
ual morphs, which are different from those of M. dawsoniorum and M. ratticaudae.
Temperature is a major factor affecting plant disease epidemics. In recent
years, tar spot disease of C. zizanioides has become increasingly prevalent in
Guangdong Province, China, especially in hot and rainy summers. Thus, the ef-
fect of temperature on the growth rate of M. chrysopogonis in vitro was eval-
uated in this study. There were no signicant differences in the minimum and
optimum growth temperatures amongst the three isolates and the optimum
growth temperature was 30 °C (Fig. 6). Research revealed that the highest
growth rate of M. paspali occurred at 25–28 °C and M. majus, M. seminicola
and M. nivale strains in Russia and Europe grew optimally at 20–25 °C (Doohan
et al. 2003; Gagkaeva et al. 2020), while M. nivale from Slovakia grew better
at temperatures below 20 °C (Hudec and Muchová 2010). Thus, the optimum
growth temperature varies amongst Microdochium species.
A previous study showed that P. herbarum could initially induce leaf spots
and blight on vetiver grass, causing round or irregular dark brown spots, which
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Xiang Lu et al.: New taxa of Microdochium from China
are similar to the symptoms on M. chrysopogonis (Zhang et al. 2017). However,
the symptoms on M. chrysopogonis were different from those on P. herbarum in
the later period. Specically, P. herbarum caused fusiform or irregular with red-
dish-brown margins on the host plant, whereas M. chrysopogonis caused sh-
eye necrotic haloes surrounding the spot lesions on leaves. Additionally, the
disease incidences were different. P. herbarum affected 26% to 42% of plants,
while M. chrysopogonis showed a 100% disease incidence. Given the high dis-
ease incidence associated with M. chrysopogonis and its induction of leaf spots
on vetiver grass, as well as the identication of this new species, it is imperative
to conduct future studies addressing the host spectrum, epidemic conditions,
biological characteristics and distribution patterns of M. chrysopogonis.
The effectiveness of biofungicides, such as bacterial seed treatments using
Pseudomonas and Pantoea in controlling diseases caused by Microdochium,
has been established (Johansson et al. 2003). However, there remains sub-
stantial reliance on registered chemical fungicides. Currently, research on
fungicide sensitivity within Microdochium mainly focuses on three species:
M. panattonianum, M. majus and M. nivale. Six groups of fungicides, namely,
MBCs, DMIs, QoIs, SDHIs, PPs, and dicarboximides, have been shown to have
signicant inhibitory activity (Kaneko and Ishii 2009; Aamlid et al. 2017, 2018;
Matušinsky et al. 2017; Gagkaeva et al. 2022). In this study, consistent with pre-
vious ndings, four DMI fungicides (difenoconazole, propiconazole, usilazole
and tebuconazole) and one MBC fungicide (carbendazim) exhibited signicant
inhibitory effects on the growth of M. chrysopogonis, with mean EC50 values of
0.077, 0.011, 0.004, 0.024 and 0.007 μg/ml, respectively (Table 4). However, di-
carboximides (iprodione), which were effective against snow mould and Micro-
dochium patch caused by M. nivale on turf-grass in previous studies (Gourlie
and Hsiang 2017), showed ineffectiveness in this study, with a mean EC50 value
of 193.031 μg/ml. Additionally, while the PP fungicide udioxonil demonstrated
good antifungal activity against M. majus in other research (Mao et al. 2023),
the isolates in this study displayed only moderate sensitivity to udioxonil, with
an EC50 value of 4.525 μg/ml and complete inhibition of mycelial growth re-
quired concentrations exceeding 100 µg/ml (Table 4, Fig. 8). These variations
in fungicide sensitivity could be attributed to genetic structural changes, intro-
ducing bias in chemical control ecacy (Matušinsky et al. 2019). Furthermore,
the response of the same pathogen to fungicides can vary amongst regions.
For example, the DMI fungicides, tebuconazole and metconazole, were report-
ed to be ineffective against M. nivale in the Czech Republic and France (Ioos
et al. 2005; Matušinsky et al. 2019). Similarly, M. nivale exhibited sensitivity to
SDHI fungicides, including pydiumetofen, uxapyroxad and penthiopyrad, in
vitro, but these fungicides were ineffective in providing acceptable control un-
der eld conditions in the USA (Hockemeyer and Koch 2022). These differences
may be attributed to variations in environmental factors, such as temperature
and humidity, as well as diverse biological characteristics, including epidemiol-
ogy, fungicide sensitivity and aggressive nature of the pathogen (Abdelhalim et
al. 2020). Overall, this study offers valuable insights into fungicide application
strategies for effectively managing the disease. Further research is needed to
analyse the inuences of environmental variables and conduct eld trials to
validate the effects of DMI fungicides, ultimately enhancing the ability to suc-
cessfully manage vetiver leaf tar spot disease.
225
MycoKeys 100: 205–232 (2023), DOI: 10.3897/mycokeys.100.112128
Xiang Lu et al.: New taxa of Microdochium from China
Acknowledgements
We thank the Herbarium of the Chinese Academy of Forestry for helping with
the preservation of plant specimens.
Additional information
Conict of interest
The authors have declared that no competing interests exist.
Ethical statement
No ethical statement was reported.
Funding
This research was nancially supported by the Guangdong Basic and Applied Basic Re-
search Foundation (2020A1515110167), School-level Talents Project of Lingnan Nor-
mal University (ZL 2034), Natural Science Foundation of Guangdong Province, China
(2023A1515011676) and Key Scientic Research Platform and Project of Guangdong
Education Department (2021KCXTD054)
Author contributions
Xiang Lu, Xue-Li Niu and Wu Zhang carried out the investigation and sampling. Xiang Lu and
Wu Zhang conducted the morphological and phylogenetic analysis. Xiang Lu and Wu Zhang
carried out the pathogenicity test. Xiang Lu, Meng-Xian Mai, Wen-Hui Tan, Mu-Yan Zhang,
Jie Xie and Yi Lu undertook the fungicide sensitivity experiment. Xiang Lu wrote, edited and
reviewed the manuscript. Xiang Lu and Wu Zhang reviewed the manuscript and provided
funding. All authors have read and agreed to the published version of the manuscript.
Author ORCIDs
Xiang Lu https://orcid.org/0000-0001-9582-1319
Mengxian Mai https://orcid.org/0009-0001-3824-2895
Wenhui Tan https://orcid.org/0009-0008-6054-2174
Muyan Zhang https://orcid.org/0009-0005-8880-9780
Jie Xie https://orcid.org/0009-0002-4200-0140
Yi Lu https://orcid.org/0009-0003-0848-2390
Data availability
All of the data that support the ndings of this study are available in the main text or
Supplementary Information.
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231
MycoKeys 100: 205–232 (2023), DOI: 10.3897/mycokeys.100.112128
Xiang Lu et al.: New taxa of Microdochium from China
Supplementary material 1
Phylogenetic tree inferred from a maximum likelihood analysis based on a
combined alignment of ITS sequences of 97 isolates of the Microdochium sp.
Authors: Xiang Lu, Mengxian Mai, Wenhui Tan, Muyan Zhang, Jie Xie, Yi Lu, Xue Li Niu,
Wu Zhang
Data type: tif
Explanation note: Bootstrap support values obtained with ML above 70% and Bayesian
(BI) posterior probability values above 0.90 are shown at the nodes (BI/ML). The des-
ignated outgroup taxa are Thamnomyces dendroidea CBS 123578. Numbers of ex-
type strains are emphasized with an asterisk and species are delimited with shaded
blocks. Isolates of M. chrysopogonis are indicated with lighter text.
Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.
Link: https://doi.org/10.3897/mycokeys.100.112128.suppl1
Supplementary material 2
Phylogenetic tree inferred from a maximum likelihood analysis based on a
combined alignment of LSU sequences of 72 isolates of the Microdochium sp.
Authors: Xiang Lu, Mengxian Mai, Wenhui Tan, Muyan Zhang, Jie Xie, Yi Lu, Xue Li Niu,
Wu Zhang
Data type: tif
Explanation note: Bootstrap support values obtained with ML above 70% and Bayesian
(BI) posterior probability values above 0.90 are shown at the nodes (BI/ML). The des-
ignated outgroup taxa are Thamnomyces dendroidea CBS 123578. Numbers of ex-
type strains are emphasized with an asterisk and species are delimited with shaded
blocks. Isolates of M. chrysopogonis are indicated with lighter text.
Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.
Link: https://doi.org/10.3897/mycokeys.100.112128.suppl2
232
MycoKeys 100: 205–232 (2023), DOI: 10.3897/mycokeys.100.112128
Xiang Lu et al.: New taxa of Microdochium from China
Supplementary material 3
Phylogenetic tree inferred from a maximum likelihood analysis based on a
combined alignment of rpb2 sequences of 71 isolates of the Microdochium sp.
Authors: Xiang Lu, Mengxian Mai, Wenhui Tan, Muyan Zhang, Jie Xie, Yi Lu, Xue Li Niu,
Wu Zhang
Data type: tif
Explanation note: Bootstrap support values obtained with ML above 70% and Bayesian
(BI) posterior probability values above 0.90 are shown at the nodes (BI/ML). The des-
ignated outgroup taxa are Thamnomyces dendroidea CBS 123578. Numbers of ex-
type strains are emphasized with an asterisk and species are delimited with shaded
blocks. Isolates of M. chrysopogonis are indicated with lighter text.
Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.
Link: https://doi.org/10.3897/mycokeys.100.112128.suppl3
Supplementary material 4
Phylogenetic tree inferred from a maximum likelihood analysis based on a
combined alignment of tub2 sequences of 80 isolates of the Microdochium sp.
Authors: Xiang Lu, Mengxian Mai, Wenhui Tan, Muyan Zhang, Jie Xie, Yi Lu, Xue Li Niu,
Wu Zhang
Data type: tif
Explanation note: Bootstrap support values obtained with ML above 70% and Bayesian
(BI) posterior probability values above 0.90 are shown at the nodes (BI/ML). The des-
ignated outgroup taxa are Thamnomyces dendroidea CBS 123578. Numbers of ex-
type strains are emphasized with an asterisk and species are delimited with shaded
blocks. Isolates of M. chrysopogonis are indicated with lighter text.
Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.
Link: https://doi.org/10.3897/mycokeys.100.112128.suppl4
Available via license: CC BY 4.0
Content may be subject to copyright.
