The document describes an ultrastructural study of cells of Phytophthora infestans, the pathogen that causes late blight in tomatoes and potatoes, after treatment with the fungicide oxadixyl. The study found that in some hyphae and haustoria of P. infestans in infected tomato leaves treated with oxadixyl, there was massive accumulation of electron-dense deposits near the plasma membrane and mitochondria. This led to mitochondrial degeneration and likely cell death. However, no changes were observed in the endoplasmic reticulum or number of ribosomes. Some hyphal cell walls were also thickened after oxadixyl treatment. The damage to fungal cells from oxadix

1. J. Phytopathology 132, 116—128 (1991)
© 1991 Paul Parey Scientific Publishers, Berlin and Hamburg
ISSN 0931-1785
Universitat Hohenheim, Institut fur Phytomedizin
Postfach 700562, 7000 Stuttgart 70, Federal Republic of Germany
Cellular Damage to Phytophthora infestans in Tomato Leaves
Treated with Oxadixyl: an Ultrastructural Investigation
YoNG JIANG and F. GROSSMANN
Authors' addresses: Y. JIANG, Department of Crop Protection, Waite Agricultural Research Institute,
Glen Osmond, SA 5064 (Australia). F. GROSSMANN, Universitat Hohenheim, Institut fiir Phytomedi-
zin (360), Postfach 700562, D-7000 Stuttgart 70 (Fed. Rep. of Germany).
With 12 figures
Received March 14, 1990; accepted January 9, 1991
Abstract
Ukrastructural observations showed massive accumulation of electron dense deposits adjacent
to plasmalemma and mitochondria and mitochondrial degeneration, which probably leads to cell
death, in some hyphae and haustoria of Phytophthora infestans in infected tomato leaves treated with
the systemic fungicide oxadixyl (8 ;Ug/ml). However, no changes in the endoplasmic reticulum and
number of ribosomes were observed. The cell walls of some hyphae were markedly thickened after
oxadixyl treatment. Degenerated haustoria were partly present in intact host cells. Evidently, fungal
cell damage by treatment with oxadixyl is not correlated with host cell damage.
Zusammenfassung
Zellschaden bei Phytophthora infestans in Tomatenblattern
nach Behandlung mit Oxadixyl: Eine ultrastrukturelle Untersuchung
Ultrastrukturelle Untersuchungen zeigten eine massive Akkumulation von elektronendichten
Ablagerungen in unmittelbarer Nachbarschaft des Plasmalemmas und der Mitochondrien sowie
Degeneration der Mitochondrien, die wahrscheinlich zum Zelltod fiihrt, in einigen Hyphen und
Haustorien von Phytophthora infestans in infizierten Tomatenblattern, die mit dem systemischen
Fungizid Oxadixyl (8 Mg/ml) behandelt worden waren. Veranderungen des endoplasmatischen Reticu-
lums und der Zahl der Ribosomen wurden jedoch nicht beobachtet. Ferner waren die Zellwande
einiger Hyphen nach der Behandlung betrachtlich verdickt. Degenerierte Haustorien wurden teil-
weise in intakten Wirtszellen gefunden. Offensichtlich korreliert die Schadigung der pilzlichen Zellen
nach Behandlung mit Oxydixyl nicht mit der Schadigung der Wirtszellen.
U.S. Copyright Clearance Center Code Statement: 0931-1785/91/3202-0116$02.50/0

2. Cellular Damage to Phytophthora infestans Treated with Oxadixyl 117
Oxadixyl is a systemic fungicide acting mainly on the Peronosporales of
oomycetous fungi (FULLER and Gisi 1985). Based on the similarity of the chemical
structure and the cross-resistance to metalaxyl, a systemic fungicide in use since
many years, it is expected that oxadixyl may have the same inhibitory effect on
rRNA synthesis of Oomycetes as metalaxyl (COHEN and COFFEY 1986, DAVIDSE
et al. 1988). Ukrastructural studies revealed that ribosomes of Phytophthora
infestans were indeed reduced after treatment with the fungicide (unpublished).
Moreover, the haustorium development of the fungus was also inhibited (unpub-
lished) and interpreted as the consequence of the fungistatic effect. However,
application of oxadixyl in combination with the protective fungicide, mancozeb,
successfully controls plant diseases caused by pathogens which are even resistant
to metalaxyl (Gisi et al. 1985) and oxadixyl displays less activity in vitro than, but
the same activity in vivo as, metalaxyl (COHEN and COFFEY 1986). Therefore,
differences between oxadixyl and metalaxyl in their mode of action seem to exist.
In an ukrastructural study with Phytophthora infestans in infected tomato
leaves treated with oxadixyl, it was observed that oxadixyl exhibited not only
fungistatic, but also fungitoxic effects on the fungus, which have not yet been
reported on metalaxyl-treated fungi in vitro and in vivo (HiCKY and COFFEY
1980, GROHMANN and HOFFMANN 1982, COHEN and COFFEY 1986). Hence, the
possibility of another action site of oxadixyl is implied to be present.
Materials and Methods
Fungus material
Phytophthora infestans Mont (de Bary), race 1.4, was cultured on potato discs (cv. 'Bintje') at
20 °C. Four days after inoculation, sporangia were collected in distilled water and incubated at 12 °C
for 3—4 h until zoospores were released. The resulting zoospore suspension was used for inoculation
of leaves.
Plant material
Seedlings of Lycopersicum esculentum (cv. 'Bonny Best'), susceptible to the above mentioned
race, were planted in 9-cm-diameter plastic pots containing a mixture of Humosoil, perlite and sand
(1 : 1 : 1, v/v) and cultivated in the greenhouse at 18—26 °C. Five-week-old plants were used for the
experiments.
Fungicide and application
Oxadixyl [2-methoxy-N-(2-oxo-l,3-oxazolidin-3-yl)acet-2',6'xylidide], technical grade, 96 %
pure, was obtained from Sandoz Ltd. It was dissolved in tap water (8 jug/ml, equal to the EC50 value)
and sprayed onto the upper surfaces of tomato leaves 24 h before inoculation. As a control, only water
was sprayed.
Inoculation
Small filter paper discs (5 mm in diameter) were dipped in a freshly prepared zoospore
suspension (10* ml"') and placed on the lower surfaces of the third and fourth leaves, in order to avoid
direct contact between the fungicide and the fungus before infection. After inoculation, all plants were
kept in a moist chamber at 20 °C.
Electron microscopy
15 and 18 h after inoculation, the inoculated areas of the leaves were excised with a razor blade
and placed in buffered glutaraldehyde (3 % in 0.6 mol/1 cacodyiate, pH 7.0, containing 3 mmol/1
l), fixed at room temperature for 2 h. After washing (3 x 20 tnin) in the same buffer, the

3. 118 JIANG and GROSSMANN
specimens were postfixed m 2 % unbuffered OSO4 for 2 h. Dehydration was carried out in a graded
ethanol series (max. 70 %) followed by simultaneous staining overnight at 10 °C in 5 % uranyl
acetate. After further dehydration, the probes were embedded in Spurr's medium. Ultrathin sections
1
Fig. 1. Mature hypha of P. infestans showing its organelles such as the ellipsoid-shaped nucleus (n),
mitochondria (m) with tubular cristae, the endoplasmic reticulum (ER, arrow) and the dictyosome (d)
connected with the nuclear membrane etc. (Control, 34 000 X, at 18 h)

4. Cellular Damage to Phytophthora infestans Treated with Oxadixyl 119
were made randomly from all five embedded blocks of the same treatment with a microtome
(Reichert-Jung) and a diamond knife. Sections were stained with uranyl acetate (5 % in 10 % ethanol
at room temperature for 50 min) and lead citrate (20 min), and then observed using a Zeiss EM 9
(60 kV).
Micrographs from the ultrathin sections were generally magnified 20 000 X. Number of
ribosomes was counted in an area of 0.25 cm^ randomly selected in the micrographs and then
converted into the number in the authentic area of 1 jum^. Data were analyzed statistically.
Results
Appearances of mature cells of P. infestans
Similar to the observations made by SHIMONY and FRIEND (1975), cells of
P. infestans in tomato leaves could be divided into three developmental stages:
young, mature and senescent. At the mature stage, the endoplasmic reticulum
(ER) was attached to ribosomes and appeared as double-layered linear structures
in thin sections (Fig. 1). Dictyosomes were connected either with the ER or the
nuclear envelope. Ribosomes were distributed evenly in the cytosol and were also
attached to the outer nuclear membranes, with their number ranging around
680 ± 67 (mean ± SD)//im^. The nucleus in the hyphae appeared ellipsoid, with
the nuclear pores and nucleolus recognizable. Mitochondria contained tubular
cristae. At this stage, vacuoles occurred in different size in the cells. Of the total
thin sections (presented as 550 micrographs) of the fungus serving as control by
15 and 18 h after inoculation, about 80 % showed that matured appearances,
10 % the senescence.
Appearances of senescent cells of P. infestans
The senescent fungal cells of the control were observed, mainly, in the outer
spongy mesophyll at both 15 and 18 h after inoculation. The senescent process
could be divided further into four stages based on the extent to which the
Fig. 2. Hypha at the beginning of the senescence. The ER has swollen (arrow), ribosomes became
sparce and accumulated in groups in the cytoplasm {e.g., in white ovals). The nucleus is globular.
(Control, 20 000 X, at 18 h)

5. 120 JIANG and GROSSMANN
austorium in the middle stage of the senescence. Electron dense deposits appear at iFig. 3. A haustorium in the middle stage of the senescence. Electron dense deposits appear at nuclear
(n) and mitochondrial (m) membranes and at tonoplasts of some vacuoles. The host cell has
degenerated already, showing no extrahaustorial membrane. (Control, 20 000 X, at 15 h)
ukrastructural changes in organelles, especially mitochondria, occurred in this
study: 1. Primary stage of the senescence; appearance of many organelles except
mitochondria were changed. The ER was fragmented and swelled, but remained
attached to ribosomes, which, however, were not in contact with the nuclear
envelope. The nucleus did not show its fine structure and became globular. In the
cytosol, the number of ribosomes decreased by 25 ± 14 % (P < 0.05 after
Student's test) compared to the mature cells. Most of the ribosomes accumulated
together as groups, probably as polyribosomes (Fig. 2). Vacuoles appeared as
many small ones or as an enlarged central one. However, in some sections,
vacuoles were not evident due to presence of other organelles. 2. Middle stage of
the senescence; amorphous electron dense material was deposited at mitochon-
drial membrane, nuclear envelope and tonoplasts (Fig. 3). The ribosomes were no
longer evident. The tonoplasts disappeared partly. 3. Late stage of the senescence;
mitochondrial cristae disappeared to a great extent or completely, whereas the ER
was more dilated but maintained the membrane structure (Fig. 4). Meanwhile,
nuclear membranes, especially the inner ones, partly disappeared. The tonoplasts
disappeared completely. However, the plasma membrane remained intact. 4.
Final stage of the senescence; all organelles became unrecognizable (Fig. 5),

6. Cellular Damage to Phytophthora infestans Treated with Oxadixyl 121
• ••••:'.• • •• ':
' ' l .
•• A
Fig. 4. A hypha in the late stage of senescence. Mitochondrial (m) cristae have disappeared. The ER is
more dilated. Tonoplasts of most vacuoles (v) become degenerated. Only plasmalemma remains
intact. (Control, 20 000 X, at 15 h)
Fig. 5. Collapsed cytoplasm of a haustorium without recognizable organelles. The host cell possesses
no visible structure except the remaining rest of the extrahaustorial membranes (arrows). (Control,
20 000 X, at 18 h)

7. 122 JIANG and GROSSMANN
indicating the thorough loss of the compartmentation. The collapsed cytoplasm
appeared electron dense and then very electron lucent. It is noteworthy of
mentioning that the host cells containing the senescent haustoria had already
collapsed (Fig. 5).
: ; • ; - : : . • ^ ; ; - ; . . ^ • . = • - / • - • • • > ; ; • ^ • ^ • • : . ' i ' ^ ^ . .
' ! " - • ' - " . - " • ' . - ' - . • ! • . " > , / i ' ^ / - " ' , ^ ; ? . • - • • • • . ' ' " • ' " . • . ' - . . ' " • . ; • V > - . • ' : . • • , ' ' • . • - . • • '
, - ' , ' : v • • • • . ; ' " • ; • " ; • -• ' • • ' . • ' • ' ' - / ' • ' ' l ^ " , . : O W - - ^ ' ' '
: " ' • V / , • . • . - , " r i ; ; ; . ' . • , - • . ; • • . . . • • ' . . . • . . : • . : i . ; • - . > v / . • . .
Fig. 6. Hypha after treatment with oxadixyl. Some electron dense deposits are visible at plasmalemma
(arrows), whereas the ER, dictyosome and mitochondria are not changed. (Oxadixyl treatment,
20 000 X, at 15 h)
^" « i " . * " ' . " - i " ' . ' ; • - ' • " " - . ' • ' " ' ' ' ' - • : • * . - • ' ' • ' ' . ' " ' * ' * *' ' ^ ' * ' * , - * ' ' : • : ' * * . • , . • . * - '
Fig. 7. The electron dense deposits develop extensively from the plasmalemma to some region of the
cytoplasm. The plasmalemma (arrows) is located outside the electron dense deposits. Mitochondria
(m) have degenerated and are coated by the deposits as well. The ER remains as double-layered linear
structure despite its attachment to some deposits. (Oxadixyl treatment, 20 000 X, at 15 h)

8. Cellular Damage to Phytophthora infestans Treated with Oxadixyl 123
Appearances of damaged cells of P. infestans after treatment with oxadixyl
By 15 and 18 h after inoculation, the treatment with oxadixyl caused striking
differences in the appearance of the fungal cells. In addition to other observations
demonstrated as fungistatic effects (unpublished), also about 10 % of the thin
sections (presented as 570 micrographs) made from the same plant tissue in the
Fig. 8. Similar to Fig. 7. The ER and the dictyosome seem not to be injured and separated. The
vacuoles are still electron lucent, indicating intact tonoplasts. Ribosomes are sill attached to the ER
and not reduced. (Oxadixyl treatment, 20 000 X, at 15 h)
G -k-^^"-'
:C:'^y.:^.^:>-Z'-':o::
• . • •
^., t. 'f.'-' '
,.-VS5»=~
Fig. 9. The ER has not changed its ultrastructural appearance (arrows) although the nucleus (n) and
the mitochondria (m) degenerated evidently. The nuclear envelope has disappeared. (Oxadixyl
treatment, 20 000 X, at 18 h)

9. 124 JIANG and CROSSMANN
oxadixyl treatment, exhibited the following changes which were characterized by
the accumulation of electron dense deposits in the fungal cells. The electron dense
deposits occurred near or at the plasma membranes and/or at mitochondrial
membranes (Fig. 6—7). At the same time, however, the ER did not change and
the distribution pattern and number (about 640 ± 81/jdxn^) of ribosomes showed
no significant difference from those of the mature cells of the control. Mitochon-
dria became degenerated during the time of accumulation of the electron dense
deposits (Fig. 7 and 9), with the fine structure of the cristae lost. With the time,
the electron dense deposits accumulated massively in the cytosol near the plasma
membrane and extended towards the central area of the cell (Fig. 7—8). The
plasma membrane itself did not become electron dense (Fig. 7), but could be
covered by the massive deposits. During the process, changes in the fine structure
of the nuclei could not be observed. However, the nuclear membranes dis-
appeared and were also in contact with the electron dense deposits (Fig. 9). The
whole nucleus degenerated. Interestingly, the ER remained still unchanged or less
modified, even in spite of attachment to some electron dense deposits. The
ribosomes were recognizable. Finally, the collapsed cytoplasm was electron dense
and/or electron lucent, with the plasmalemma and the ER lost. The phenomenon
described here occurred occasionally also in small haustoria, together with the
degenerated mother hyphae. In addition, degenerated haustoria were evidently
present in intact host cells (Fig. 10).
Fig. 10. A degenerated haustorium with the unchanged ER and collapsed mitochondria (m) present in
the host cell which shows the intact mitochondrion, tonoplasts and chloroplasts. (Oxadixyl treatment,
20 000 X, at 18 h)

10. Cellular Damage to Phytophthora infestans Treated with Oxadixyl 125
Fig. 11. Thickened cell walls of a hypha. The cytoplasm is filled with some vacuoles. (Oxadixyl
treatment, 7000 X, at 18 h)
In some intercellular hyphae, cell walls were thickened considerably
(Fig. 11). These cells generally showed the seemingly fragmented but not dilated
ER with ribosomes. In addition, vacuolation of the cells was also very conspicu-
ous. However, electron dense deposits were seldom observed. It seemed that the
• - ' . . • , ' :
Fig. 12. Some cytoplasmic vesicles are fusing with the plasmalemma of the hypha (arrows), probably
leading to the thickening of the cell walls. Vacuoles are evident in the cytoplasm. (Oxadixyl treatment,
20 000 X, at 18 h)

11. 126 JIANG and GROSSMANN
thickening of the cell walls was due to the accumulation of the cytoplasmic,
probably dictyosome, vesicles near the cell walls and their fusion with the
plasmalemma (Fig. 12).
Discussion
The results showed that in both oxadixyl treatment and the untreated control
there were appearances of damaged cell organelles of P. infestans due to the
fungicide treatment and the physiological senescence, respectively. Hence, it is
necessary to clarify the differences between the mature and senescent cells in the
control and the cells affected by the fungicide to draw conclusions here.
The senescent process occurring in P. infestans is mainly timed as reduction
in ribosomes, dilation of the ER, degeneration of mitochondria and plas-
maiemma. Changes in ribosomes and ER may reflected increased activity of
ribonucleases and proteolysis (MATILE 1971, GAHAN 1981). It seems likely that
changes in the ER and appearance of the electron dense deposits are ultrastruc-
tural markers of the senescence, whereas the degeneration of mitochondria is the
marker in the senescent process leading to death, as described in irreversible
events occurring in necrosis of animal cells (WYLLIE 1981). Degeneration of
mitochondria and instability of the mitochondrial genome are closely correlated
with senescence of fungi (MUNKRES 1985). In many cases, the dead cells of higher
plants are finally characterized by irreversible plasmolysis, disruption of cellular
membranes with a concomitant loss of compartmentation, loss of respiratory
activity etc. (GAHAN 1981). Occurrence of one of these features indicates that the
cell is dead. Evidently, the degenerated mitochondria may have no respiratory
activity and present the irreversible "switch on" of the step in cell senescence
leading to cell death. Thus, any change in the cells after mitochondrial degenera-
tion could only be a post-mortem event. It was considered that loss of compart-
mentation of cells can be an indicator of the moment of cell death (MATILE 1975).
But it is more likely that cell death has occurred before the loss of compartmenta-
tion (GAHAN 1981). This seems to be the case here, i.e., the cell death might occur
after degeneration of mitochondria but before disappearance of the plasmalemma,
especially in treatment with oxadixyl.
The genetically programmed cell senescence (MUNKRES 1985, 1987) and cell
death of P. infestans seems to be strikingly different from the cellular damage
caused by treatment with oxadixyl. Although the ultrastructural appearance of
the fungus in oxadixyl treatment is similar to that of the untreated control at the
late stage, i.e., mitochondrial degeneration and degeneration of plasmalemma, the
ER, the possible marker of the senescence, surprisingly remains unchanged even
after mitochondrial degeneration. The most notable difference lies in the exten-
sive occurrence of the electron dense deposits and their location at plasmalemma.
The deposits here are similar to the cytoplasmic densities observed in this and
other fungi after treatment with other fungicides (MULLER and BuRTH 1983,
HOCH and SYKOLNIK 1979). Presumably, the deposits adjacent to the plas-
malemma and in mitochondria could be denatured proteins (WYLLIE 1981) which
were formed m involvement of the fungal plasmalemma and mitochondrial
membranes.

12. Cellular Damage to Phytophthora infestans Treated with Oxadixyl 127
It is not known whether the occurrence of the cell damage of the type as
described above only in a minority of the fungal cells (10 % in all thin sections) is
due to a relatively high concentration of the fungicide encountered by the fungus
in vivo or to the high sensitivity of these fungal cells. The latter seems to be more
possible in that different appearances of the fungus such as cell wall thickening
and fungitoxic phenomena occur in different hyphae located in the same plant
tissue. In vivo and in vitro, many oomycetous fungi show different sensitivity to
metalaxyl in the same species (COFFEY and BOWER 1984, COFFEY et al. 1984, COOK
and ZHANG 1985, HERZOG and SCHOEPP 1985, HUNGER et al. 1982, SHEW 1984),
dependent on the growth environment (FULLER and Gisi 1985). Additionally,
with P. infestans, isolates are frequently found that are resistant to metalaxyl in
vitro and yet highly sensitive to it in vivo (COHEN and COFFEY 1986). This could
be due to the induction of plant resistance by metalaxyl (BORNER et al. 1981,
CAHILL and WARD 1989). However, with oxadixyl, another possibility seems to
be the fungitoxic action on some hyphae which become sensitive to oxadixyl after
infection of the host plants. Further investigations are needed to support the
suggestion.
The work was supported by the Deutsche Gesellschaft fiir Technische Zusammenarbeit. Mrs.
G. MOLL developed part of the photographs.
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