8 ff3 fs7 fc0 sc0 ls2 ws4">virulence factors of the phytopathogenic organisms that
produce them [23-25]. Despite these findings, there are
few reports on the effects of these mycotoxins at the ul-
trastructural level in plants. Ultrastructural analysis and
cytological methods have been very useful in identifying
the effects of other compounds designed to injure plants
(synthetic herbicides), and some of these results are
paramount to determining and understanding the uptake
and translocation, and the molecular mode of action of
such compounds in plant tissues.
The primary mode of trichothecene action in eu-
karyotic cells is inhibition of the synthesis of protein,
DNA, and RNA [26,27]. Phytotoxicity of trichothecenes
was first reported in the early 1960s, e.g., [28]. Later,
other trichothecenes were found to be phytotoxic, e.g.,
one report [27] found 15 trichothecenes with phytotoxic
activity and that the macrocyclic tricothecenes were more
potent. Ultrastructural analysis of some Stachybotrys
chartarum isolates with or without the gene producing
satratoxin (macrocylic trichothecene) have been reported
Even though substantial information has been amassed
concerning the utility of MV as a bioherbicide, little is
known about its infectivity mechanism(s) and virulence
factors. Thus the objectives of the present experiments
were to examine the ultrastructural effects of high purity
roridin A and MV spores on kudzu tissues early after
application to seedlings, in order to glean more precise
information on the action of this marcocyclic trichothe-
cene alone, compared to that of MV spores. This would
provide important information related to the primary or
initial events of MV infection or disease manifestation,
and aid in the elucidation of possible mechanisms of ac-
tion of MV and of one of its metabolites (roridin A) on
host plant organelles and structural constituents.
2. Materials and Methods
2.1. Inoculum Production
Myrothecium verrucaria (MV) spores (IMI 361690)
were grown in Petri dishes containing potato dextrose
agar (PDA). The plates were incubated (28˚C, fluores-
cently-lighted incubator, 7 - 10 days) and spores were
washed from the agar surfaces with sterile distilled water
and filtered through two layers of cheesecloth to remove
clumps of agar. The concentration of the spore suspen-
sion thus obtained was adjusted using a hemacytometer
to yield an inoculm containing 1 × 106 spores·ml–1. The
surfactant Silwet L-77 (Silwet) (Osi Specialties, Inc.;
Charlotte, NC, USA) was added to the final inoculum to
attain a concentration of 0.20% (v/v).
2.2. Test Plant Propagation
Kudzu seeds (Adams-Briscoe Seed Co., Jackson, GA
30233, USA) were placed on moistened filter paper in
Petri dishes, and incubated at 28˚C for 3 days in the dark.
Germinated seeds were then planted in 7.6 cm plastic
pots (one seed per pot) containing a 1:1 commercial pot-
ting mix (Jiffy Products of America, Inc., Batavia, IL
60510, USA): sandy loam soil combination, supple-
mented with a controlled-release (13:13:13, N:P:K) fer-
tilizer (Grace Sierra Horticultural Products, Milpitas, CA
95035, USA). After placement on greenhouse benches,
the plants were sub-irrigated daily. Greenhouse tempera-
tures ranged from 28˚C - 32˚C at 40% - 60% RH with a
photoperiod of about 14 h, at 1600 - 1800 µmol·m–2·s–1
PAR as measured at midday.
2.3. Test Plant Inoculation
Kudzu seedlings at the 1 - 2 leaf stage, grown as de-
scribed above, were inoculated by brushing (small art-
ist’s brush) with Silwet, MV plus Silwet, or roridin A at
10–4 M (Sigma Chemical Co, St. Louis, MO) prepared in
Silwet. The treated seedlings were then placed in a dew
chamber (28˚C, 12 h), and incubated in a greenhouse as
previously described above. Leaf and stem tissues were
collected at 1 to 48 h after treatment (HAT). Control
plants received 0.2% Silwet surfactant only. Each repli-
cate contained five seedlings, with three replications.
2.4. Electron Microscopy
The kudzu tissues were excised at the appropriate sample
times after treatment, and several seedlings from each
treatment were harvested at each time point. The tissues
sections were fixed (6% glutaraldehyde), post-fixed (2%
osmium tetroxide), stained (2% uranyl acetate), dehy-
drated (acetone), embedded (epoxy resin), sectioned with
Copyright © 2012 SciRes. AJPS
Effects of Myrothecium verru caria on Ultrastructural Integrity of Kudzu
(Pueraria montana var. lobata) and Phytotoxin Implications
a Reichert UltraCut E microtome (Vienna, Austria), and
examined using a Zeiss EM10CR transmission electron
3. Results and Discussion
3.1. Disease Symptomology at the
Ultrastructural Level
3.1.1. Visual Symptoms
Treatment of kudzu seedling leaflets with MV spore
preparations resulted in disease symptomatology charac-
terized by necrotic flecking which occurred within 2 h
following treatment. Disease symptoms progressed from
the cotyledons and leaves producing stem lesions within
about 48 h (data not shown).
3.1.2. Ultrastructural Symptoms
At the ultrastructural level, MV treatment caused rapid
effects associated with detachment of the protoplast from
cell walls, rapidly resulting in cell death. There were no
visual or ultrastructural effects caused by MV spores
alone, or by the surfactant Silwet alone in sections from 6
and from 24 h after treatment (Figure 1). All organelles
are well preserved and the tonoplast and plasmamem-
brane are intact. In fact, the ultrastructral preservation
obtained from the Silwet samples was actually superior
to the untreated controls (not shown) probably because of
the facilitation of the penetration of the fixatives into the
plant tissue (Figure 1). Previous studies have indicated a
lack of infectivity by MV unless the surfactant Silwet
was used [5,6].
There was a rapid (~1 - 6 HAT) protoplast detachment
from cell walls (Figure 2), and although the cells were
mostly intact, small perturbations were evident at the
wall-plasma membrane interface. By 12 HAT (Figure 3),
the cell at the top of the figure is affected only slightly
more than the ones after 6 HAT (Figure 2), while the
Silwet Control
Figure 1. Electron micrograph of kudzu mesophyll cells
from seedlings treated with 0.2 % Silwet L-77 (control), 6
HAT. Vc = vacuole; PM = plasma membrane; T = tono-
cell at the bottom (Figure 3) has nearly separated its
plasma membrane from the cell wall. As a consequence,
the plasmodesmata has been stretched. Also many plas-
modesmata appeared to be broken off and retained in cell
walls (not shown). It should be noted that all of these
ultrastructural symptoms occurred prior to the appear-
ance of fungal growth structures. However, fungal
growth was observed following severe tissue degenera-
tion (24 to 48 HAT), but growth occurred primarily on
the leaf surfaces, rather than within the plant tissue (not
shown). There was also a complete displacement of
plasma membranes from the wall surfaces and distortion
of many of the cells (Figure 4).
Roridin A in surfactant treatment caused symptoms
similar to those induced by spores formulated in surfac-
tant (Figure 5(A)). This trichothecene is present in un-
washed MV spores, but can be removed by washing [30].
In contrast to the treatment with MV spores alone, treat-
ment of kudzu leaves with roridin A gives only patchy
perturbations of cell structure. In this low magnification
electron micrograph (Figure 5(A)), the cell in the center
and the 2 adjacent cells in the bottom-left quadrant have
lost their tonoplasts and their cellular contents are dis-
Figure 2. Electron micrograph of kudzu cells 6 HAT of M.
verrucaria spores in Silwet L-77 (0.2%). V = vesicle; PM =
plasma membrane.
MV, 12 HAT
Figure 3. Electron micrograph of kudzu mesophyll cells
treated with M. verrucaria spores in Silwet L-77 (0.2%), 12
HAT. PD = plasmodesmata; PM = plasma membrane.
Copyright © 2012 SciRes. AJPS
Effects of Myrothecium verru caria on Ultrastructural Integrity of Kudzu
(Pueraria montana var. lobata) and Phytotoxin Implications
MV, 24 HAT
Figure 4. Electron micrograph of kudzu mesophyll cells
treated with M. verrucaria spores in Silwet L-77 (0.2%), 24
HAT. PM = plasma membrane.
Roridin A, 24 HAT
Roridin A, 24 HAT
Figure 5. Electron micrograph of kudzu leaf tissue, 24 HAT
with roridin A (104 M) in Silwet L-77 (0.2%). (A) = rela-
tively low magnification showing patchy effects occurring
on only a few cells; (B) = largest patch of cells found to be
affected in roridin A treated leaf sections examined.
rupted. However, most of the remaining cells in the sec-
tion, are apparently only moderately affected or unaf-
Kudzu treated with roridin A 24 HAT (Figure 5(B))
also exhibited detachment of the plasma membrane from
the cell wall. This is the largest patch of cells found to be
affected in any roridin A treated leaf sections examined
and even they are flanked by essentially unaffected cells
in the upper-right and bottom-left quadrants. This local-
ized damage may indicate poor translocation or move-
ment of this mycotoxin within the plant tissues. Previous
research in our laboratory has suggested that roridin A is
not rapidly translocated in kudzu seedlings [20].
Treatment with the MV toxin roridin A plus Silwet
however gave a number of unique effects upon the tissue.
Most striking was the apparent detachment of the proto-
plast from the cell wall. At early stages of this process,
organelles within the cell appeared normal but increas-
ingly the protoplast exhibited symptoms of necrosis and
leakage of phenolics and other substances from the vacu-
ole. Plasmodesmata, that normally would connect the
protoplasts of adjacent cells are either stretched or de-
tached during this process of protoplast detachment. De-
spite this dramatic effect on the cells, there was no indi-
cation of either hyphae or spores even in these severely
disrupted cells. Thus, it is likely that this disruption is
due to some diffusible substance, perhaps a phytotoxin,
that causes the plant tissue injury, rather than to fungal
invasion of the cell.
These data suggest that penetration of a phytotoxic
substance(s) in the fungal formulation is facilitated by
the surfactant, and clearly show that the phytotoxic ac-
tion of roridin A on kudzu tissues that are similar to MV
toxin(s). Some ultrastructural studies on the effects of
non-macrocyclic and macrocyclic trichothecenes have
been published, but most have dealt with mammalian
systems, e.g., effects on mice tissues [31], myocardial
microvasculature [32], and effects on sperm motility and
changes in the plasma membrane [29]. With regard to
pathogen-crop plant interactions, the infection process of
Fusarium graminearum in wheat plants has been inves-
tigated using ultrastructural and immunocytological me-
thodologies [33]. Deoxynivalenol and its acetylated de-
rivatives are the most commonly found non-macrocyclic
trichothecenes in F. graminearum-infected plant tissues.
Scanning and transmission electron microscopy were
also used to study the infection process, the spread of
hyphae of F. culmorum, and the localization of tricho-
thecenes in wheat tissues [34]. These authors found that
the fungus developed dense mycelium on the inner plant
tissue surfaces with invasion into the lemma, glume,
palea and ovary by penetration pegs. During spreading of
the fungus, alterations in host tissues including degenera-
tion of cytoplasm, cell organelles, and deposition of elec-
tron dense material between cell wall and plasmalemma
were observed. Ultrastructural studies revealed that host
cell walls in proximity to the fungal penetration pegs,
and in contact with hyphae, were less dense or transpar-
ent suggesting involvement of cell wall degrading en-
zymes. Enzyme- and immunogold-labelling confirmed
the presence of cellulases, xylanases and pectinases. Hy-
drolytic (proteolytic) activity has been detected and as-
Copyright © 2012 SciRes. AJPS
Effects of Myrothecium verru caria on Ultrastructural Integrity of Kudzu
(Pueraria montana var. lobata) and Phytotoxin Implications
sayed in MV [35]. Localization studies also demon-
strated the presence of trichothecenes in host tissues
early after infection. F. culmorum and other Fusarium
spp. produce several trichothecenes: nivalenol, deoxyni-
valenol, fusarenon X, 3-acetyldeoxynivalenol and 15-
acetyldeoxynivalenol, and zearalenone, a macrocyclic
lactone derivative of resorcilic acid [36]. We are unaware
of any reports suggesting that F. culmorum synthesizes
macrocyclic trichothecenes as do Myrothecium spp.
However, the phytotoxicity of many non-macrocyclic tri-
chothecenes has been documented [37].
4. Conclusions
Our results are the first to report on the ultrastructure
effects of a high purity macrocyclic trichothecene (ro-
ridin A) on plant tissue. This also appears to be the first
report of ultrastructural studies of MV on plant tissues.
Overall, the results suggest that the effects of MV spores
cannot be explained solely by presence of roridin A. This
point is strongly supported by recent findings in our
laboratory that indicate washed MV spores that are void
of trichothecenes, remain highly efficacious against
kudzu [36], and that mycelia preparations of MV (also
void of trichothecenes) are as phytotoxic as unwashed
MV spore preparations when applied to plants [12].
These latter two points would tend to rule out marcocyc-
lic trichothecenes (roridin A) as a virulence factor of MV
against kudzu. The overall ultrastructural effects of MV
spores applied in Silwet also appear unique among those
caused by identified phytotoxins or mycotoxins.
Knowledge of MV virulence factors would greatly aid
the development and improvement of a commercial bio-
herbicidal product. Cytological investigations at the ul-
trastructural level will yield invaluable information on
the mechanism(s) of action of MV. Although we have
recently shown that mycelial formulations of MV are
highly efficacious in controlling kudzu, and do not pro-
duce roridin A or other trichothecenes at detectable lev-
els via HPLC analysis [19], further investigations are
necessary to assess the bioherbicidal effects of mycelial
preparations and washed spore formulations of MV on
plant tissues of various weeds. Research is also in pro-
gress to assess the role of hydrolytic enzymes in the
virulence of MV and to discover the nature of possible
non-trichothecene phytotoxins produced by MV. Ad-
vances in the understanding of bioherbicidal mechanisms
of action and the elimination of mycotoxins in MV for-
mulations will help promote this fungus as a safe and
effective bioherbicide.
5. Acknowledgements
The authors thank Andrew J. Bowling for discussion and
helpful suggestions concerning this manuscript.
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Copyright © 2012 SciRes. AJPS
Effects of <i>Myrothecium verrucaria</i> on Ultrastructural Integrity of Kudzu (<i>Pueraria montana var. lobata</i>) and Phytotoxin Implications
American Journal of Plant Sciences, 2012, 3, 1513-1519
http://dx.doi.org/10.4236/ajps.2012.311182 Published Online November 2012 (http://www.SciRP.org/journal/ajps)
Effects of Myrothecium verrucaria on Ultrastructural
Integrity of Kudzu (Pueraria montana var. lobata) and
Phytotoxin Implications
Robert E. Hoagland1, C. Douglas Boyette2, Kevin C. Vaughn1, Neal D. Teaster1, Ken Stetina2
1United States Department of Agriculture-Agricultural Research Service (USDA-ARS), Crop Production Systems Research Unit,
Stoneville, USA; 2USDA-ARS, Biological Control of Pests Research Unit, Stoneville, USA.
Email: bob.hoagland@ars.usda.gov
Received June 10th, 2012; revised September 6th, 2012; accepted October 8th, 2012
The fungus Myrothecium verrucaria (Alb. & Schwein.) (MV), originally isolated from diseased sicklepod (Senna obtu-
sifolia L.), has bioherbicial activity against kudzu and several other weeds when applied with low concentrations of the
surfactant Silwet L-77. To more fully understand the initial events of MV infection or disease progression, and to im-
prove knowledge related to its mechanism of action, the effects of MV and its product (roridin A) on kudzu seedlings
were examined at the ultrastructural level. Ultrastructural analysis of MV effects on kudzu seedlings revealed a rapid
(~1 h after treatment) detachment of the protoplast from the cell wall and plasmodesmata appeared to be broken off and
retained in the wall. These symptoms occurred well in advance of the appearance of any fungal growth structures. Some
fungal growth was observed after severe tissue degeneration (24 to 48 h after treatment), but this occurred primarily at
the extra-cellular location with respect to the kudzu tissues. Kudzu seedlings treated with roridin A, a trichothecene
produced by the fungus, exhibited some symptoms similar to those induced by the fungus applied in spore formulations
with surfactant. The overall results are the first to report the ultrastructural effects of this bioherbicide on plants and
suggest that penetration of a phytotoxic substance(s) in the fungal formulation was facilitated by the surfactant, and that
roridin A exerts phytotoxicity toward kudzu.
Keywords: Bioherbicide; Biological Weed Control; Kudzu; Myrothecium verrucaria; Ultrastructure; Trichothecene
1. Introduction
Kudzu [Pueraria lobata (Willd.) Ohwi], a perennial le-
guminous vine native to eastern Asia, was introduced
into the US in the late 1800’s [1] and now occurs from
Florida to New York, westward to central Oklahoma and
Texas, with the heaviest infestations in Alabama, Geor-
gia, and Mississippi causing over $340 million yr1 in
losses [2]. Cited in a 1993 Congressional Report as one
of the most harmful non-indigenous plants in the US,
kudzu was labeled a federal noxious weed in 1998. This
aggressive weed is very difficult to control using syn-
thetic chemical herbicides [3] and has been identified as
an over-wintering host of Asian soybean rust (Phakop-
sora pachyrhi zi Syd. & P.) [4].
Bioherbicides (microorganisms and/or their products)
can cause injury and/or mortality to weeds. One example
of a fungal bioherbicide is Myrothecium verrucaria (Alb.
& Schwein.) Ditmar:Fr., originally isolated from dis-
eased sicklepod (Senna obtusifolia L.). This wild-type M.
verrucaria (MV) exhibited excellent biocontrol potential
for several weed species, including the legumes sickle-
pod and hemp sesbania [Sesbania exaltata (Raf.) Rydb.
ex A.W. Hill] when formulated with the silicone-poly-
ether surfactant Silwet L-77 (OSi Specialties, Inc., Char-
lotte, NC) [5]. A patent for use of MV as a biological
control agent on kudzu was issued [6], and this pathogen
was found to be highly virulent to kudzu in the absence
of dew [7].
We are evaluating MV as a potential bioherbicide for
kudzu [7] and several other economically important
weeds [8-10]. MV effectively controls kudzu in the ab-
sence of a dew treatment over a wide range of physical,
environmental, and field conditions [7]. Reports indicate
significant performance improvements of several bioher-
bicides through additive or synergistic effects of chemi-
cals [11] and we reported synergistic interactions of MV
and glyphosate on kudzu and some other weeds [12-14].
MV host range tests on various tree species commonly
found in natural kudzu infestations indicated most trees
were not susceptible to MV [15].
Copyright © 2012 SciRes. AJPS
Effects of Myrothecium verru caria on Ultrastructural Integrity of Kudzu
(Pueraria montana var. lobata) and Phytotoxin Implications
Despite the positive bioherbicidal aspects of MV, one
negative factor is its production of macrocyclic tricho-
thecenes which are known to be mycotoxins [16]. Being
aware of the risks associated with this trait, we have dis-
cussed and presented the risk factors associated with
bioherbicides and outlined strategies to overcome or cir-
cumvent this problem in MV [13,17,18]. Methodologies
have been developed to isolate and quantify trichothe-
cenes produced by MV [19,20]. Several reports indicate
that some macrocyclic trichothecenes are phytotoxic and
a non-specific biological assay was developed to measure
the presence of trichothecenes based on their effects on
an alga (Chlorella vulgaris) and on two fungi (Ustilago
maydis and Trichoderma viride) [21]. The phytotoxicity
of some trichothecenes has also been examined using
another alga, Chlamydomonas reinhardtii, as a model
system [22]. Furthermore, some reports suggest that
macrocyclic and non-macrocyclic compounds act as
Effects of Myrothecium verru caria on Ultrastructural Integrity of Kudzu
(Pueraria montana var. lobata) and Phytotoxin Implications
Copyright © 2012 SciRes. AJPS
[35] R. E. Hoagland, C. D. Boyette and M. A. Weaver, “Hy-
drolytic Enzymes Produced by a Bioherbicidal Strain of
Myrothecium verrucaria,” Picogram, Vol. 73, 2007, p.
[36] A. Bottalico, “Fusarium Disease of Cereals: Species
Complex and Related Mycotoxin Profiles,” European
Journal of Plant Pathology, Vol. 80, No. 1, 1998, pp. 8-
[37] D. Masuda, M. Ishida, K. Yamaguchi, I. Yamaguchi, M.
Kimura and T. Nishiuchi, “Phytotoxic Effects of Tricho-
thecenes on the Growth and Morphology of Arabidopsis
thaliana,” Journal of Experimental Botany, Vol. 58, No.
7, 2007, pp. 1617-1626. doi:10.1093/jxb/erl298