Effects of <i>Myrothecium verrucaria</i> on Ultrastructural Integrity of Kudzu (<i>Pueraria montana var. lobata</i>) and Phytotoxin Implications

doi:10.4236/ajps.2012.311182

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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)

1513

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: [email protected]

Received June 10th, 2012; revised September 6th, 2012; accepted October 8th, 2012

ABSTRACT

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 yr−1 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].

Effects of Myrothecium verru caria on Ultrastructural Integrity of Kudzu

(Pueraria montana var. lobata) and Phytotoxin Implications

1514

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

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

[29].

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

Effects of Myrothecium verru caria on Ultrastructural Integrity of Kudzu

(Pueraria montana var. lobata) and Phytotoxin Implications

1515

a Reichert UltraCut E microtome (Vienna, Austria), and

examined using a Zeiss EM10CR transmission electron

microscope.

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-

plast.

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-

MV, 6 HAT

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.

Effects of Myrothecium verru caria on Ultrastructural Integrity of Kudzu

(Pueraria montana var. lobata) and Phytotoxin Implications

1516

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

Figure 5. Electron micrograph of kudzu leaf tissue, 24 HAT

with roridin A (10−4 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-

fected.

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-

Effects of Myrothecium verru caria on Ultrastructural Integrity of Kudzu

(Pueraria montana var. lobata) and Phytotoxin Implications

1517

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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