Plant Science 175 (2008) 818–825
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Plant Science
journal homepage: www.elsevier.com/locate/plantsci
Phyllostictine A, a potential natural herbicide produced by Phyllosticta cirsii:
In vitro production and toxicity
Maria Chiara Zonno a,*, Maurizio Vurro a, Sergio Lucretti b, Anna Andolfi c,
Carmen Perrone c, Antonio Evidente c
a
b
c
Institute of Sciences of Food Production, National Research Council, via Amendola 122/O, 70125 Bari, Italy
Plant Genetics and Genomics Section, Biotechnologies, Agro-industries and Health Protection Department, ENEA Casaccia Research Centre, Via Anguillarese 301, 00123 Rome, Italy
Department of Soil, Plant, Environmental and Animal Production Sciences, University of Naples Federico II, via Università 100, 80055 Portici, Italy
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 7 May 2008
Received in revised form 31 July 2008
Accepted 5 August 2008
Available online 19 August 2008
Phyllostictine A is a powerful toxin produced by Phyllosticta cirsii, a potential mycoherbicide of Cirsium
arvense. To support its potential use as a natural herbicide, toxin production has been studied using
different media and cultural conditions. The toxin content in the crude extracts has been determined by
using a HPLC method set up for this purpose. Furthermore, its phytotoxicity has been evaluated on
tobacco protoplasts by flow cytometric analysis, and on C. arvense protoplasts, by fluorescence
microscopy. The best cultural conditions found allowed to produce more than 28 mg ml 1 of toxin in
culture filtrate. The pure metabolite proved to have rapid dose-dependant toxic effects on host and nohost plant protoplasts.
ß 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords:
Phyllostictine A
Flow cytometry
Cirsium arvense
Nicotiana tabacum
HPLC analytical method
Protoplasts
1. Introduction
Weed pathogens are considered interesting potential sources of
novel natural herbicides. Recently Phyllosticta cirsii has been
proposed as a potential mycoherbicide for the biological control of
the noxious and widespread weed, Cirsium arvense, commonly
known as Canada thistle [1]. Species belonging to the genus
Phyllosticta are known to produce bioactive metabolites, including
non-host phytotoxins, e.g.: phyllosinol, brefeldin and PM-toxin,
isolated by cultures of Phyllosticta sp., P. maydis and P. medicaginis,
respectively [2–4]. This led us to investigate the production of
toxins by P. cirsii [5]. From the fungal liquid culture phyllostictine A
(1, Fig. 1) was purified and chemically characterized as a new
oxazatricycloalkenone. This toxin, isolated together with other 3
related metabolites (named phyllostictines B–D) (2–4, Fig. 1), in
preliminary bioassays showed interesting biological properties. In
particular, it proved to have a noteworthy phytotoxicity, no
antifungal activity and an antibiotic and zootoxic activity at high
concentrations. Considering its interesting and promising properties, phyllostictine A was proposed as a potential natural herbicide
* Corresponding author. Tel.: +39 080 5929332; fax: +39 080 5929374.
E-mail address: mariachiara.zonno@ispa.cnr.it (M.C. Zonno).
0168-9452/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2008.08.003
deserving further studies on its phytotoxicity. Three main
objectives were defined for the present study: (a) finding the best
conditions for phyllostictine A in vitro production, in order to
obtain sufficient amounts of the toxin to perform the biological
assays, and to evaluate the capability of the fungus to produce this
metabolite in vitro; (b) setting up a rapid analytical method to
determine the phyllostictine A content in the culture filtrates, in
order to easily compare the influence of the different cultural
conditions on the toxin production; (c) assaying the phytotoxic
activity of phyllostictine A, to better evaluate its potential as
natural herbicide.
2. Material and methods
2.1. Optimizing fungal growth and phyllostictin A production
A strain of Phyllosticta cirsii was supplied by Alexander
Berestetskiy, Russian Research Institute of Plant Protection,
Saint-Petersburg, Russia. It was stored in the mycological
collection of Institute of Science of Food Production (ITEM N.
8964) in 20% glycerol at 80 8C. For inoculum production, it was
transferred to potato-dextrose-agar (PDA) plates and grown at
25 8C under near UV lights for 2 weeks in order to obtain a
sufficient production of picnidia. Plates were then used to prepare
M.C. Zonno et al. / Plant Science 175 (2008) 818–825
819
Fig. 1. Structures of phyllosticnines A–D (1–4).
conidial suspensions. In order to find the best conditions for fungal
growth and toxin production, the fungus was grown in four
mineral liquid media, namely Malt extract [6], Fries modified [7],
M-1-D [8] and Kent-Strobel [9]. For each medium, both Erlenmeyer
flasks (containing 400 ml of medium) and Roux bottles (containing
200 ml medium) were used, for shaken and static conditions,
respectively.
Aliquots of suspensions (approximately 106 conidia ml 1) were
used to inoculate flasks and bottles (0.1 and 0.2 ml respectively)
that were then incubated in shaken (100 rpm) or static conditions,
respectively, at the dark and at 25 8C.
Static cultures were harvested at week intervals, up to 9 weeks,
whereas the shaken cultures were harvested at four-day intervals,
up to 20 days. All the cultures were produced in duplicate.
The fungus was also grown on M-1-D medium with reduced
saccharose content (1/2, 1/5 or 1/10 of the standard recipe, i.e.
28.25 g l 1), or with glucose (28.25 g l 1) as carbon source, or even
with the addition to the standard M-1-D medium [10] of minced
fresh leaves of C. arvense (2 g l 1), collected from naturally infested
fields near Bari. These inoculated media were incubated for 7
weeks in static condition at 25 8C at the dark. After incubation, the
cultures were filtered on filter paper (Whatman N.4), mycelium
fresh and dry weights were determined and pH of culture filtrates
was measured. All culture filtrates were tested for the toxicity by
using the leaf puncture assay described below. Aliquots (100 ml)
were lyophilized and stored until the determination of the
phyllostictin A content, as described below.
2.2. Leaf puncture bioassay
To compare the biological activity of the culture filtrate to the
phyllostictine A content, the toxicity of all the cultured filtrates
obtained under various conditions and media was tested by a
puncture assay on thistle leaves. Young leaves were cut from C.
arvense plants grown in the greenhouse, obtained from seeds
collected from wild plants in a field near Bari.
Droplets (20 ml) of solutions were placed on punctured leaves
that were kept in a moist chamber at room temperature for 72 h.
The diameter of the necrotic area was measured.
2.3. HPLC analysis—toxin content
The lyophilised fungal culture filtrates (corresponding to 50 ml
for the static cultures and to 100 ml for the shaken cultures),
obtained as reported above, were extracted tree times by CHCl3–
iso-propanol (9:1, v/v) solution (15 and 30 ml for static and shaken
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M.C. Zonno et al. / Plant Science 175 (2008) 818–825
cultures respectively). The organic phases were combined, filtrated
on paper (Whatman N.4), and evaporated under reduced pressure.
The samples were dissolved in methanol prior to their use.
The phyllostictine A standard sample was purified and
identified from P. cirsii culture filtrates as described in a previous
work [5]. The HPLC calibration curve for quantitative phyllostictine
A determination was performed with standard amounts of the
pure toxin dissolved in methanol in the range between 0.05 and
20 mg ml 1 and each concentration was run in triplicate. HPLC
linear regression curve (absolute amount against chromatographic
peak area) for phyllostictine A was obtained as regression
weighted lines calculated from nine multiple injections of the
standard metabolite in the concentration range above indicated.
For the determination of the phyllostictine A content in the
fungal cultures, aliquots of the samples (20 ml) were injected for
analysis, having the mobile phase consisting in acetonitrile and
HPLC grade water (1:1, v/v), and with a flow rate of 1 ml min 1.
Detection was performed at 263 nm, corresponding to the
maximum phyllostictine A absorption [5].
Each sample analysis was in duplicate. The quantitative
determination of the metabolite was calculated interpolating
the mean area of their chromatographic peaks with the data of the
calibration curves.
concentrations [12]. Furthermore, the herbicide Glyphosate (trade
name: Glifone, 360 g l 1 of pure active compound) was tested at
four concentrations, i.e.: the technical dose (t.d.: 500 l ha 1
corresponding to 4.2 10 3 M) and three decimal dilutions
corresponding to 1/10, 1/100 and 1/1000 of the t.d.
2.7. Cirsium protoplasts
Aliquots (1 ml) of protoplast suspensions containing the
metabolites at the desired concentrations were placed in
Eppendorf tubes and incubated for 1, 3 and 6 h at 27 8C at the
dark. After incubation, 50 ml of the protoplast suspension was
added with 5 ml of fluorescine diacetate (FDA: stock 1 mg ml 1)
and further incubated for 5 min. Droplets (15 ml) of treated cell
suspensions were transferred onto a microscope slide, and the
viability of cells Cell viability was assessed by using an epifluorescence microscope (Leitz, Orthoplan). Cells with intact
plasma membranes fluoresced bright green, while dead cells
remained unstained or weakly yellow-green. Methanol solutions
(1%) were also used as control to assess cellular toxicity of the
solvent. All assays were repeated in triplicate. Toxicity was
expressed as protoplast viability percentage as: Number of green
fluorescent protoplasts/Total number of protoplasts 100 and
each sample was compared to the untreated control.
2.4. Recovery studies
2.8. Tobacco protoplasts
Recovery studies were performed using the filtrate obtained by
the 2-week-old fungal culture on M-1-D medium. Pure phyllostictine A was added to the culture filtrate in the range between 0.3
and 2.0 mg l 1. The samples were managed as described above and
the extracts analysed by the HPLC method in order to determine
the recovery values. Three replicate injections were performed for
each concentration.
2.5. Protoplast bioassays
Tobacco leaf protoplast suspension (3.6 ml) were placed in
sterile 5 ml polystyrene round-bottom tubes (Falcon 2254, Becton
Dickinson, Franklin Lakes, NJ USA) and incubated as described
before. Protoplast suspension (400 ml) was stained with
0.1 mg ml 1 FDA and incubated for 7 min in polystyrene tubes.
The toxicity was evaluated by using a FACSCalibur flow cytometer
(Beckton-Dickinson, USA), by recording the green fluorescein
emission (FL1) collected with a 530/42 nm band pass filter and red
chlorophyll auto-fluorescence (FL2) (670/13 nm band pass filter).
Data were recorded and analysed using the software CellQUEST 3.1
(Becton Dickinson, USA). Viable leaf protoplasts were characterized by simultaneous emission of green and red fluorescence, FDA
and chlorophyll emission, respectively. Five thousands events
were collected for each analysis at a flow rate of 100 cells s 1.
Assays were repeated three times and all concentrations were
tested in triplicate. Toxicity was expressed as percentage of viable
protoplasts and compared to the control.
2.5.1. Protoplasts extraction
For the production of protoplasts, plants of C. arvense were
grown in soil in plastic pots (12 cm diameter) by sowing seeds
harvested in naturally infested fields near Bari, Italy. Nicotiana
tabacum cv. Samsung plants were grown on Murashige and Skoog
Basal Medium (Sigma) agar (1.2% of agar) medium in sterile
Magenta boxes (Sigma, 100 ml medium box 1). Plants were grown
with a photoperiod of 16 h light at 25 8C (lamps OSRAM HQIT
400 W/N/SI), for 1 month (thistle) or for 8 weeks (tobacco).
Leaf tissues were cut with a razor blade into small strips (about
1mm wide) and placed in Petri dishes containing 10 ml of an
enzymatic solution made of Cellulase ‘‘Onozuka’’ R-10 (Yakult,
Honsha Co. Ltd, Nigashi Shimbashi, Tokyo, Japan) (0.2%, w/v)
Driselase (0.025%, Sigma), Macerozyme R-10 (0.025%, Yakult
Honsha Co. Ltd, Nigashi Shimbashi, Tokyo, Japan) and D-Mannitol
(0.5 M, Sigma–Aldrich), CaCl2H2O (10 mM, Carlo Erba Analyticals)
and MES (3 mM, Sigma–Aldrich). Suspensions were put on an
orbital shaker overnight at 27 8C, then filtrated through a cell
strainer (70 mm) and re-suspended twice in the holding buffer
after centrifugation at 50 g for 10 min. Protoplasts were cultured
in the buffer solution at a final concentration of 105 protoplasts ml 1 [11].
2.8.1. Chemicals and equipments
Analytical and HPLC grade solvents for chromatographic use
were acquired from Carlo Erba (Milan, Italy). All other analytical
grade chemicals were purchased from Merck (Darmstadt, Germany). Water was HPLC quality, purified in a Milli-Q system
(Millipore, Bedford, MA, USA).
The HPLC system employed was supplied by Shimatzu (Tokio,
Japan) and consisted of a Series LC-10AdVP pump, FCV-10AlVP
valves, SPD-10AVVP spectrophotometric detector and DGU-14A
degasser. The HPLC separations were performed using a MachereyNagel (Duren, Germany) high-density reversed-phase Nucleosil
100-5 C18 HD column (250 mm 4.6 mm i.d.; 5 mm) provided
with an in-line guard column from Alltech (Sedriano, Italy).
2.6. Protoplast bioassay
3. Results
Protoplasts were exposed at 5 different concentrations of
phyllostictine A, i.e.: 10 3, 5 10 4; 10 4; 5 10 5 and 10 5 M).
For comparative studies, the fungal toxin fusaric acid (Sigma–
Aldrich) dissolved in methanol (1%) was tested at the same
3.1. Fungal growth
If the dry weight of the P. cirsii mycelium is considered, the best
medium for the fungal growth proved to be the malt extract, both
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Table 1
Toxicity of the culture filtrates obtained by growing the fungus on different media
in static conditions
Weeks
Media
M-1-D
1
2
3
4
5
6
7
8
9
+
+++
++++
+++
+++
++
Fries
++
+
+
+
+
Malt
Kent-Strobel
+
+
+
+
+
+
+
+
+
+
+
The assay was performed on punctured thistle leaves. Scale of toxicity: , nontoxic; +, necrosis 1 mm; ++, 2–3 mm; +++, 3–5 mm; ++++, wider necrosis.
Table 2
Toxicity of the culture filtrates obtained by growing the fungus on different media
in shaken conditions
Days
Media
M-1-D
4
8
12
16
20
Fries
Malt
Kent-Strobel
+
–
–
–
–
++
+++
+++
+
+
+
+
+
The assay was performed on punctured thistle leaves. Scale of toxicity: , nontoxic; +, necrosis 1 mm; ++, 2–3 mm; +++, 3–5 mm; ++++, wider necrosis.
3.2. Leaf puncture bioassay
Fig. 2. Growth of Phyllosticta cirsii on different media in static conditions up to 9
weeks. Changes in fungal dry and fresh weights and pH of the culture filtrates.
After 72 h incubation, the strongest toxicity was observed on
leaves treated with M-1-D culture filtrates from 4 to 7 weeks
(Table 1, static culture). A lower phytotoxic activity was caused by
younger or older M-1-D filtrates, whereas an almost negligible or
nil toxicity was observed all the way for the other three media used
(Table 1).
In shaken conditions, phytotoxic activity was also observed for
the culture filtrate obtained growing P. cirsii on Fries medium,
whereas negligible was the toxicity for the other three media used
(Table 2).
3.3. Toxin quantification
in shaken (data not shown) and static (Fig. 2) conditions. For this
latter, both fresh and dry weights increased during the whole
experiment, reaching more than 34 g and almost 3.5 g flask 1,
respectively. Intermediate results were obtained using Fries or M1-D medium (Fig. 2), whereas the worst medium proved to be
Kent-Strobel medium for both conditions, with an almost
negligible growth. With regard to the pH of the culture filtrates,
in the static conditions it was quite stable for M-1-D and Malt,
ranging between 4.8 and 5.8, whereas it was more variable for the
other two media.
With regard to the method set up for the quantification of the
phyllostictine A content in the culture filtrates, the characteristics
of the calibration curves, the absolute amount range and the
detection limits (LOD) of phyllostictine A are reported in Table 3.
The linearity of the calibration curves in the reported interval is
proven by the regression data.
The best elution conditions to quantify phyllostictine A content
in the extract from culture filtrates of P. cirsii were obtained on C18 reverse phase using isocratic elution with acetonitrile and HPLC
grade water (1:1, v/v) with a flow rate of 1 ml min 1. HPLC
Table 3
Analytical characteristics of calibration curvea for phyllostictine A
tR (min)
Range (mg)
Slope
Intercept
S.D. (y%)
r2
Number of data point
Limit of detection (LOD) (ng)
13.8
0.05–20
9153.6
917.7
0.64
0.9977
27
4.8
tR: retention time.
a
Calculated in the form y = a + bx where y is chromatographic peak area and x is mg of toxin.
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Fig. 3. HPLC profile of phyllostictine A standard peak (1) (A) and CHCl3–iso-propanol extract of P. cirsii culture filtrate (B).
chromatographic profiles of the standard phyllostictine A (A) and
of the CHCl3–iso-propanol extract of P. cirsii culture filtrate (B) are
reported in Fig. 3. In samples the chromatographic peak (1) of
phyllostictine A was identified by the coincidence of the retention
time with that of the standard, which was eluted at 13.8 min with a
high reproducibility (0.50 min). For all the samples, the matrix
substances absorbing at 263 nm were eluted within the first 6 min.
This finding, together with the high similarity of the features of the
metabolite peak in samples with those of standard, allow to indicate
that no peaks of other substances overlap those investigated.
Injecting phyllostictine A in amounts higher than the upper
limit of the indicated ranges it was still possible to have a linear
response. Amounts below the lower limit were still detectable but
with a low reproducibility of peak integration, which highly
increased the standard deviation of the regression curves.
As shown in Table 4, the content of phyllostictine A in the
culture filtrates obtained using different cultural conditions ranged
between 0.02 and 28.8 mg l 1.
Phyllostictine A was produced with a linear increase since the
first week (2.2 mg l 1) until the fourth, when it reached the
maximum (28.8 mg l 1). After that, the production linearly
decreased until the seventh week (15.2 mg l 1). In Table 4, the
fungal growth kinetic data are shown up to 9 weeks in static
condition onto M-1-D medium.
A similar trend was obtained also for the other two media, Fries
and Malt, but the production of phyllostictine A was much lower
than M-1-D. It linearly increased since the first week (0.08 and
0.04 mg l 1, for Fries and Malt, respectively), reached the maximum at the fourth week (0.59 and 0.96 mg l 1, for Fries and Malt,
respectively), and decreased linearly until the ninth week (0.22
and 0.02 mg l 1, for Fries and Malt, respectively). Onto KentStrobel medium the fungus did not produce the toxin in any of the
conditions used (data not shown).
Under shaken conditions on M-1-D, Fries and Malt media the
fungus produced phyllostictine A only after 4 days and in very low
amounts, and only in the latter medium (1.46 mg l 1), whereas no
production was detected for longer incubation times, irrespective
the medium used (data not shown).
The production of phyllostictine A using glucose as carbon
source gave a toxin yield (7.9 mg l 1) lower than saccharose. The
production of phyllostictine A strongly decreased when using
lower amounts of saccharose in the medium, lowering to 4.83, 1.75
Table 4
Production of phyllostictine A by Phyllosticta cirsii grown on different mediaa in
static conditions up to 9 weeks
Weeks
1
2
3
4
5
6
7
8
9
a
Toxin content (mg l
1
)
M-1-D
Fries
Malt
2.12
3.59
13.72
28.83
23.69
21.93
18.18
16.91
15.23
0.04
0.05
0.08
0.96
0.52
0.22
0.11
0.06
0.02
0.08
0.15
0.35
0.59
0.38
0.36
0.30
0.26
0.22
M-1-D, fries, and malt are different media.
M.C. Zonno et al. / Plant Science 175 (2008) 818–825
and 0.05 mg l 1 for the production in M-1-D medium containing 1/
2, 1/5 and 1/10 of the standard recipe saccharose content,
respectively (data not shown). A slight increase in phyllostictine
A production was obtained in 7-week-old cultures by the addition
of C. arvense leaves (21.9 mg l 1 compared to 19.1 mg l 1 of the
standard culture).
3.4. Recovery studies
The recovery of phyllostictine A added at various amounts to
the culture filtrate ranged between 94 3.5 and 97 2.1% (data not
shown). These results also indicated that the extraction by CHCl3:isopropanol (9:1, v/v) was a satisfying procedure for quantitative
analysis of the metabolite in the culture filtrates.
823
3.5. Thistle protoplasts assay
In the assay on thistle protoplasts, at the highest concentration
assayed (10 3 M) phyllostictine A was strongly effective already
1 h after its application, being able to kill almost the totality of
protoplasts (Fig. 4). At 5 10 4 M the toxicity was time dependant,
being mortality around 50% 1 h after the treatment and reaching
100% after 6 h. At lower concentrations the toxicity of phyllostictine was low even after 6 h, being almost completely inactive at
10 5 M. With regard to fusaric acid used for comparison, it proved
to have a time-dependant toxicity at the highest concentration
tested (10 3 M) (Fig. 4), causing around 60% mortality 1 h after the
application, reaching 100% about after 6 h. The other treatments
were sensibly less toxic, and not dependant by the time of
exposure. In case of glyphosate, it proved to be highly toxic at the
t.d. causing the total mortality of thistle protoplasts 3 h after the
treatment, whereas at 1.10 and 1:100 dilutions it caused around
60% protoplast mortality after 3 h of exposure to the toxin. At the
lower dose (1/1000 t.d.) it caused a noteworthy (60%) protoplast
mortality only 6 h after the treatment.
3.6. Tobacco protoplasts assay
In Fig. 5, fluorescence emissions from tobacco protoplasts
treated with phyllostictine A and analysed by flow cytometry, are
shown. Damaged protoplasts showed less and less fluorescent
Fig. 4. Toxicity of phyllostictine A (upper) fusaric acid (middle) and glyphosate
(lower) at different concentration assayed on thistle protoplasts (% of viability
compared to the control). Error bars indicate standard deviations of the
measurements for the three replications.
Fig. 5. Citogram (dot-plot) of tobacco protoplast vitality exposed to phyllostictine A
at 10 3 M (upper) and to methanol (1%) as control (lower) for 240 min. Quadrants:
UR + LR = Live protoplasts UL + LL = Debris and dead cells.
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M.C. Zonno et al. / Plant Science 175 (2008) 818–825
emissions corresponding cell membrane damage (green fluorescence after FDA staining) and subsequent chloroplast releasing
into the medium (red fluorescence from chlorophyll) At the highest
concentration tested (10 3 M) phyllostictine A acted very quickly.
In fact, already 1 h after the treatment the toxin caused the death of
almost all the protoplasts (Fig. 6). At 5 10 4 M the toxin was still
very fast acting, causing around 80% mortality already 1 h after the
treatment. At 10 4 M the toxicity was time dependant, being
considerable (around 60%) after 3 h of exposure. At the lowest
concentration tested (10 5 M) there was no detectable toxic
activity (Fig. 6). With regard to fusaric acid, it proved to be highly
Fig. 6. Tobacco protoplast viability (% of the control) after treatment with
phyllostictine A (upper), fusaric acid (middle) and glyphosate (lower) at different
concentration. Error bars indicate standard deviations of the measurements for the
three replications.
toxic al 10 3 and 5 10 4 M, causing almost 100% mortality after
6 h, whereas at 10 5 M around 60–70% protoplasts remained alive
after 6 h of exposure. Glyphosate was highly toxic assayed at t.d.,
whereas proved to be much less toxic at the reduced doses assayed
(Fig. 6).
4. Discussion
One of the problems in studying and using bioactive
metabolites produced by plant pathogens is that they are often
produced and released in the culture medium in very low amounts,
making the purification and identification very difficult to be
performed. Beside the natural capability of the microbes to
biosynthesize the compounds, the cultural conditions can strongly
influence their production in vitro. In our study, the use of M-1-D
medium, with a chemically defined composition, and the growth of
the fungus in static conditions for 4 weeks proved to be the most
effective cultural condition, allowing a production higher than
28 mg l 1.
Our data showed that toxin production was not related to the
fungal growth. In fact, on malt extract medium the fungus grew
very well, producing abundant mycelium, but toxin production
was very low.
When growing the fungus for more than 4 weeks we observed a
progressive reduction in the toxin content, which is probably due
to the instability of phyllostictine (Table 4).
The toxicity of the culture filtrates seems to be correlated to the
toxin content, being the most phytotoxic culture filtrates (i.e.:
static M-1-D cultures between 4 and 7 weeks) (Table 1) also those
with the highest toxin content (Table 4). Anyway, the presence of
other known (e.g. phyllostictine B, C and D already described) [5] or
novel phytotoxins cannot be excluded. This possibility is much
more evident in the case of the shaken conditions, in which a quite
interesting phytotoxicity was observed for the cultures on Fries
medium, that contains low or nil amounts of phyllostictine A.
The influence of the cultural conditions and medium types on
toxin production is well known from many plant pathogens. For
example, the production of the phytotoxic b-nitropropionic acid
by Septoria cirsii was increased by the addition of thistle leaves to
the M-1-D medium [13]. Ascochyta sonchii, a leaf pathogen of
Sonchus arvensis, produce a large amount of ascosonchine
(8 mg l 1) when grown on M-1-D static culture for 8 weeks, but
less than 1 mg l 1 when grown in shaken culture [14]. Ascochyta
rabiei produces solanapyrones A,B,C only if Czapek-Dox medium
was added with extract of host plant seeds [15,16]. Moreover, in
shaken culture A. rabiei produces only 0.1–10% of the solanapyrones produced in static culture [17].
The HPLC method for the qualitative and quantitative analysis
of phyllostictine A proved to be efficient, rapid and sensitive. This
method proved to be very useful for the identification of the best
cultural conditions for toxin production, and could also be used for
comparing the ability of different fungal strains to produce
phyllostictine A. Furthermore, it could be a valuable tool for
mycoherbicide studies. One of the main problems in using fungi as
mycoherbicides is the assessment of the strain virulence, which is
highly time and space consuming. If there were a positive
correlation between strain virulence and production of toxins, it
could be easier to select the most aggressive strains simply
selecting the best toxin producers. Furthermore the method could
also be useful to evaluate the toxin yield in the large scale
productions.
Compared to the use of whole plants, bioassays performed on
isolated protoplasts can offer some advantages, e.g. the use of
lower amounts of toxins, a shorter time for the analysis and clearer
host responses at the cellular level.
M.C. Zonno et al. / Plant Science 175 (2008) 818–825
Flow cytometry can produce information about protoplast
size, chlorophyll auto-fluorescence and fluorescence due uptake
and subsequent esterase cleavage of FDA into the cytoplasm,
therefore producing the strong green fluorescent compound
fluorescein [18]. Normally, this fluorochrome remains into the
protoplasm since its a polar compound and cannot cross freely
the cell membrane. If the membrane loses somehow its integrity
fluorescein can diffuse out of the cell which then is no more
fluorescent. Flow cytometry generates data which categorize
intact protoplasts emitting both red chlorophyll auto-fluorescence and green fluorescein and cellular debris and released
organelles. Analysis of the auto-fluorescence of unstained
tobacco protoplasts produce histograms containing debris and
dead cells (LL and LR) and red emitting mesophyll protoplasts
(UL); after staining with FDA another population was apparent
(UR). The latter population was made of active protoplasts
exhibiting both high chlorophyll auto-fluorescence level and
fluorescine fluorescence from FDA. Only protoplasts were
analyzed whereas chloroplasts and debris were excluded from
the analysis. Viable protoplasts had bright fluorescence during
flow cytometric analysis, whereas dead protoplasts were nonfluorescent (Fig. 5). Auto-fluorescence of the protoplasts not
treated with phyllostictin A was used as experimental control to
monitor the integrity of protoplast preparations combined with
FDA staining.
In our bioassays on protoplasts, at the highest concentration
used phyllostictine proved to act faster then glyphosate at the
t.d. (Fig. 4). This strong activity, together with the appearance of
necrosis when applied to leaves, could support the idea of using
the toxin as a herbicide for foliar application and tissue
desiccation. Compared to FA, which is a well known and
powerful toxin, phyllostictine proved to be much more potent
(Fig. 4).
Acknowledgements
This work was carried out within the project ‘‘Enhancement and
Exploitation of Soil Biocontrol Agents for Bio-Constraint Management in Crops’’ (contract no. FOOD-CT-2003-001687), which was
financially supported by the European Commission within the 6th
FP of RTD, Thematic Priority 5—Food Quality and Safety. The
research was also in part supported by a grant from Regione
Campania L.R. 5/02. Contribution DISSPAPA N. 158. The authors
thank Dr. Elisabetta Sbisà, Dr. Apollonia Tullo and Dr. Beatriz
825
Navarro, Institute of Biomedical Technologies, CNR, Bari, Italy, for
the assistance in managing cell bioassays.
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