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Report Phase I
Silicon nutrition and resistance against Pythium aphanidermatum of Lycopersicon esculentum and Mormodica charantia
Summary
Vegetable production in greenhouses is threatened by root rot caused by Pythium species, among them P. aphanidermatum
which is a soil-borne plant pathogen with mainly tropical distribution.
The objective of our studies was to clarify the role of Si in the
resistance of tomato against Pythium. For comparison we conducted
studies with bitter gourd (Momordica charantina) belonging to the Cucubitaceae, known to react positively to Si, on four levels: Si nutrition with particular emphasis on roots, Si/Pythium interaction in substrates on growth under controlled conditions, Si/Pythium interaction on tomato yield in the greenhouse in Thailand, and infection and development of Pythium on a single root basis.
Si nutrition
On the basis of the Si concentrations of shoots,
roots and xylem exudates it could be confirmed that tomato belongs to
the Si excluders and bitter gourd to the Si accumulator plants. As a
consequence Si accumulated in the root water free space (WFS) in tomato
whereas Si depletion in the WFS could be shown in bitter gourd. The
fractionated extraction of Si from root compartments clearly revealed
that in tomato roots, most of the Si was bound in the cell walls
whereas in bitter gourd, Si accumulated particularly in the cytoplasmic
fraction.
Si/Pythium interaction in peat
Tomato and bitter gourd seedlings were grown in a
limed peat substrate with and without inoculation by mycelium infected
soil and minus and plus Si application supplemented by a soluble Si source (Aerosil, Degussa) in the substrate. The reaction of tomato seedlings to Pythium was
very variable ranging from rapid damping off to no effect depending on
the inoculation level but independent of Si nutrition. In contrast, in
bitter goard which was more resistant to Pythium, a clear positive effect of Si could be shown.
Long-term experiment in Thailand
Three long-term experiments with
tomato were conducted in cooperation with I.Wanwilai, KU, in the
nethouse in Thailand with factorial combination of Si or Ca amendment
of a local peat substrate, Pythium infection (mycelium-infected soil), and an optimal und supra-optimal N nutrition. In both experiments, Pythium
infection slightly but significantly reduced growth of tomato plants
independent of the N nutrition. Si supply tended to exert a positive
effect on some growth parameters.
Si/Pythium interaction at the single root level
A newly developed experimental setup that facilitates
the treatment of defined root zones with particular solutions was used
to study the spread of Pythium in individual roots of tomato
and bitter gourd as affected by the zone of inoculation and by Si
supply. Inoculation was done with zoospores and after harvest the level
of Pythium colonisation in root segments was quantified by a specific ELISA. For both species, the level of Pythium
infection was highest when inoculating the root tip and a severe
infection caused cessation of root growth. In bitter gourd but not in
tomato, continuous Si supply to the complete root lead to a reduced
growth of the pathogen in the basipetal direction. In contrast,
discontinued Si supply or Si application to individual root segments
only had no effect on Pythium spread. The differences between tomato and bitter gourd to benefit from Si supply when infected with Pythium can be explained by the different distribution of Si in the roots of the species.
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Interim Report Phase II
Effect of Manganese and Silicon on the resistance of tomato (Lycopersicon esculentum Mill) against Pseudocercospora fuligena
Summary
Under the experimental conditions at the site of Bangkok, Thailand, tomato black leaf mould caused by the fungus Pseudocercospora fuligena turned
out to be the major fungal pathogen affecting tomato production. Though
chemical control is possible, ecological concerns demand environmental
friendly means of disease control.
Previous studies
revealed that manganese supply in the supraoptimal range changes the
status of the leaf apoplast via stimulation of plant peroxidase
activity and induction of PR protein expression, which could lead to an
enhanced predisposition of plants against pathogens. However, the
suspected effects of Mn supply could also be affected by leaf
application of Si, which itself was frequently reported to be a means
for the control of leaf diseases.
To clarify the
potential of Mn nutrition and Si supply for control of tomato black
leaf mold, the single as well as the combined effects of both
treatments on an artificial infection of tomato with Pseudocercospora fuligena were
studied at the experimental site of Hannover, which facilitated a
proteomic and metabolomic characterization of the underlying
biochemical processes in the leaf apoplast.
Enzyme-activity
measurements clearly demonstrated that both, moderate Mn stress and Si
supply by leaf spraying enhanced the activity of peroxidases in the
leaf apoplast of tomato. No treatment effects were found on the total
leaf area showing disease symptoms but microscopic analysis revealed
that leaf application of Si decreased the proportion of stomata
penetrated by the fungus as well as the number of fungal colonies per
unit leaf area.
When investigating
the proteome of the leaf apoplast by the means of 2-dimensional gel
electrophoresis a striking difference was found between inoculated and
control plants. For inoculated plants additional protein spots were
found and the expression of several other proteins was up regulated.
The formation of these proteins was suppressed in plants receiving
manganese treatment. A characterization by mass spectroscopy of the
respective proteins is currently in progress.
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Johannes F. J. Max, Walter J. Horst , Urbanus N. Mutwiwa, Hans-Jürgen Tantau (2007)
Effects of cooling method on greenhouse microclimate, growth, fruit yield and quality of tomato (Solanum lycopersicum L.) in a tropical climate
Submitted to Agric. For. Met.
ABSTRACT
A tomato (Solanum lycopersicum L.) crop was grown in
greenhouses (GH’s) operated either naturally ventilated (“NET”) or
evaporative cooled (“EVAP”) for 20 weeks in the dry season 2005/06 in
tropical central Thailand. Temperatures (Temp’s), relative humidity
(rH) and vapour pressure deficit (VPD) differed significantly between
the GH-types. Overall mean air Temp was reduced by 2.6 and 3.2 °C
during daytime and 1.2 and 2.3 °C at night in EVAP as compared to NET
and ambient air, respectively. Temp maxima in EVAP averaged 4.0 °C
lower than in NET and 3.9 °C lower than outside. The rH in EVAP was
increased by around 20 and 30 % during daytime and ca. 10 and 15 % at
night when compared to NET and ambient air, respectively. VPD averaged
0.25 in EVAP, 1.03 in NET and 1.48 kPa outside. The crop
water-consumption per plant and day was significantly lower in EVAP
(1.2 L) than in NET (1.8 L), which is ascribed to reduced transpiration
in EVAP, mainly resulting from lower VPD and air Temp’s. Total fruit
yield was similar in both GH-types (6.4 kg plant-1 equivalent to 96 t ha in NET and 6.3 kg plant-1 or 94.5 t ha-1
in EVAP). Although the quantity of undersized (mostly parthenocarpic)
fruits was reduced in EVAP, the overall proportion of marketable yield
was significantly higher in NET (4.5 kg plant-1, 67.5 t ha-1) than in EVAP (3.8 kg plant-1, 57 t ha-1),
owing to a largely increased incidence of Fruit Cracking in EVAP. This
coincided with increased fruit fresh weights, lower total Ca uptake and
reduced Ca concentrations in vegetative plant parts, but higher Ca
concentration in the fruits in EVAP. From the results and considering
the costs for installation and operation, it is concluded that in
regions with high ambient air rH evaporative cooling is not
recommendable without profound technical modifications.
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Johannes F. J. Max and Walter J. Horst (2007)
Effect of nighttime EC levels on growth, fruit yield and quality of tomato (Solanum lycopersicum L.) grown in greenhouses in Central Thailand
(in preparation).
ABSTRACT
Effects of two nighttime fertigation (NTF) treatments i.e. high EC (“HEC”, 3.0 mS cm-1, full solution) and low EC (“LEC”, 0.5 mS cm-1, Ca(NO3)2 and H3BO3) on the incidences of blossom-end rot (BER) and fruit cracking (FC) in greenhouse tomato (Solanum lycopersicum
L.) were investigated and compared with a control without additional
NTF (“Control”). Additional NTF cycles were applied for one hour after
the regular daytime fertigation (EC 1.5 mS cm-1). The trial
was conducted in central Thailand during the dry season (Nov. 2005 -
Mar.2006) using the two cultivars FMTT 260 and King Kong 2, known to
have high and low susceptibility for the investigated disorders,
respectively. Total amount of fruits affected by FC or BER was higher
(about 4 and 10-fold, respectively) in King Kong 2, but relatively the
reaction to the NTF-treatments was similar to FMTT 260. NTF had
significant effects on plant growth, balance of mineral elements
between supply- and leachate solution, nutrient concentrations in plant
tissue as well as the number and proportions of fruits affected by BER
and FC. HEC led to accelerated plant growth. As compared to Control and
LEC, BER-incidence in both cultivars was significantly increased in
HEC. In LEC the percentage of BER-affected fruits was lowest; though
that of cracked fruits highest. Total yield was not affected by NTF.
However, due to the induction of either BER (HEC) or FC (LEC) in both
NTF treatments the proportion of marketable yield was significantly
reduced compared to the Control. Possible reasons for the findings as
well as perspectives for the improvement of tomato fruit quality for
production in tropical climate are discussed.
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Frank Liebisch, Johannes Max, Gregor Heine and Walter J. Horst (2007)
Influence of fruit application of calcium and boron on yield and fruit quality of two tomato (Solanum lycopersicum L) cultivars grown in net houses in Central Thailand
(in preparation).
ABSTRACT
The two tomato cultivars King Kong 2 (KK2) and FMTT 260 (FMTT) were
grown in a naturally ventilated greenhouse in Central Thailand to
investigate the influence of fruit applications of combined aqueous
calcium (Ca) and boron (B) solutions amended or not with the tenside
Glycopon on fruit yield and quality. Special emphasis was laid on
blossom-end rot (BER) and fruit cracking (FC), two prevailing disorders
in tomato and main causes for non-marketability of tomato fruits. No
effect of the additional Ca and B applications on total fruit yield was
found, but the composition of the non-marketable fraction of fruit
yield was affected by treatment and cultivar. KK2 proved to be highly
susceptible to both BER and FC and, therefore, produced less marketable
fruits than FMTT. The Ca and B sprays decreased the incidence of BER
but increased FC at the same time. Addition of the tenside Glycopon to
the Ca and B solutions did not yield any further advantage. The
contrasting effects of the Ca and B sprays on BER and FC resulted in
similar levels of non-marketable fruit yields in the treatments with or
with out the sprays as well as in both cultivars. Furthermore the
nutrient status during fruit development was surveyed in different
fruit parts (proximal end, fruit pulp, side, and distal end). The
calcium content in the distal end of the fruit during the time of rapid
fruit growth was correlated to the BER incidence in the ripe fruit.
Since additional Ca and B sprays are labor-intensive and did not
increase marketable fruit yield the selection and breeding of cultivars
less sensitive of BER and FC is considered a promising strategy for
protected tomato production under tropical climate conditions.
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Heine, G., Tikum, G., Horst, W.J. (2007)
The effect of silicon on the infection by and spread of Pythium aphanidermatum in single roots of tomato and bitter gourd
J. Exp. Bot. 58, 569-577.
ABSTRACT
The effect of silicon (Si) supply on the infection and spread of Pythium aphanidermatum was studied in the roots of tomato (Solanum lycopersicum, Si excluder) and bitter gourd (Mormodica charantia,
Si intermediate accumulator). Individual roots were mounted into
polyvinyl chloride (PVC) compartmented boxes which allowed the
application of Si and zoospores to defined root zones. Two days after
inoculation root growth was recorded, and P. aphanidermatum colonization of individual root sections was determined by ELISA.
In tomato as well as in bitter gourd the root tip was the root section most sensitive to P. aphanidermatum infection. Application of Si did not affect severe root-growth inhibition by P. aphanidermatum
in either species. However, continuous Si supply significantly
inhibited the basipetal spread of the pathogen from the infected root
apex in bitter gourd but not in tomato. Si application to the roots
only during pre-treatment or only during/after the infection of the
roots failed to inhibit the spread of P. aphanidermatum.
Determination and compartmentation of Si in the roots of bitter gourd
revealed that apoplastic Si was not, but symplastic Si was associated
with the ability of the plant to reduce the spread of the fungus in
roots.
We conclude that accumulation of Si in the root cell-walls does not
represent a physical barrier to the spread of P. aphanidermatum
in bitter gourd and tomato roots. The maintenance of elevated
symplastic Si contents is a prerequisite for Si-enhanced resistance
against P. aphanidermatum.
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Heine, G.,
Moran-Puente, D.W., Horst, W.J. (2006)
Manganese and Silicon-induced changes in the proteome of the leaf
apoplast of tomato: contribution to the resistance against tomato Black
Leaf Mold
Paper presented at the International Colloquium, Plant Nutrition meets Plant Breeding, Hohenheim, Sept. 26-28, 2006.
ABSTRACT
Tomato (Lycopersicon
esculentum Mill.) production
has an enormous economic potential in SE Asia but yield in the tropics
is limited by manifold biotic and abiotic constraints. Under conditions
of protected cultivation in South Thailand, black leaf mold caused by
the fungus Pseudocercospora fuligena is the most severe fungal
tomato disease. As an
alternative to application of pesticides, strengthening of the plant
natural defense is a promising approach for disease control. It is well
known that enzymes of the antioxidative metabolism (e.g. peroxidases)
are involved in defense reactions of plants against pathogens, and the
activities of these enzymes in the apoplast can be enhanced by
supraoptimal manganese (Mn) supply (Fecht-Christoffers et al., Plant
Physiol. 133, 1935-1946, 2001). However, the role of silicon (Si) in
this context is unclear. On the one hand, leaf spraying of Si was
reported as a means to control fungal diseases (Menzies et al., J.
Amer. Soc. Hort. Sci. 117, 902-905, 1992). On the other hand, Si is
known to alleviate Mn stress (Horst et al., J. Plant Nutr. Soil Sci.
162, 263-274, 1999) which would in turn lead to a lower activity of
peroxidases. In order
to investigate the effects of Mn and Si on an infection of tomato with P. fuligena,
a factorial experiment in water culture was carried out. Mn
concentration in the solution was kept at 50 µM Mn (Mn treatment), and
leaves were daily sprayed with Si at a concentration of 7 mM (Si
treatment). Plants were grown in a mist chamber (temperature > 25°C;
humidity > 90%) thus mimicking the climatic conditions of South
Thailand. During the incubation period fungal infection was monitored
by microscopy and after appearance of visible symptoms the area under
disease was quantified. Two weeks after inoculation, leaves were
detached and apoplastic washing fluid (AWF) was extracted by using an
infiltration/centrifugation method.
Enzyme-activity measurements in the AWF clearly demonstrated that both,
Mn and Si treatments enhanced the activity of peroxidases in the leaf
apoplast. No treatment effects were found on the total leaf area
showing disease symptoms, but microscopic analysis revealed that leaf
application of Si decreased the proportion of stomata penetrated by the
fungus as well as the number of fungal colonies per unit leaf area.
When investigating the proteome of the leaf apoplast by means of
2-dimensional gel electrophoresis a striking difference was found
between inoculated and control plants. For inoculated plants additional
protein spots were found and the expression of several other proteins
was upregulated. The formation of these proteins was suppressed by Mn
treatment. A characterization of the respective proteins by mass
spectroscopy is currently in progress.
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Heine, G., G. Tikum, W. J. Horst (2006)
The effect of Silicon on the infection by and spread of Pythium aphanidermatum in single roots of tomato and bitter gourd.
Submitted to Journal of Experimental Botany.
ABSTRACT
We studied the effect of silicon (Si) supply on infection and spread of Pythium aphanidermatum
in the roots of the Si excluder tomato and the Si accumulator bitter
gourd. Individual roots were mounted into PVC compartmented boxes which
allowed the application of Si and zoospores to defined root zones. Two
days after infection root growth was recorded, and P. aphanidermatum colonization of individual root sections was determined by ELISA.
In tomato as well as in bitter gourd the root tip was the root section most sensitive to P. aphanidermatum
infection. Application of Si did not affect Pythium-induced severe
root-growth inhibition in either species. However, continuous Si supply
significantly inhibited the basipetal spread of the pathogen from the
infected root apex in bitter gourd but not in tomato. Si application to
the roots only during pre-treatment or only during/after the infection
of the roots failed to inhibit the spread of P. aphanidermatum.
Determination and compartmentation of Si in the roots of bitter gourd
revealed that apoplastic Si was not, but symplastic Si was associated
with the ability of the plant to reduce the spread of the fungus in
roots.
We conclude that accumulation of Si in the root cell-walls does not represent a physical barrier to the spread of P. aphanidermatum in
bitter gourd and tomato roots. The maintenance of elevated symplastic
Si contents is a prerequisite for Si-enhanced resistance against P. aphanidermatum.
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U. Dhakal, V. Salokhe, H.Tantau and J. Max (2005).
Development of a Greenhouse Nutrient Recycling System for Tomato Production in Humid Tropics.
Agr. Engng. Intl., 2005, Vol. VII. Manuscript BC 05 008P.
ABSTRACT
Generally it is not customary to have a nutrient recycling system for
the tropical poly-net greenhouses. The main reason is the cost
involved. However, due to recent growing environmental concerns,
efforts were made in this study to develop a simple but effective
nutrient recycling system.
Tomato (Lycopersicon esculentum Mill.,
var. FMTT-260) plants were grown under two identical poly-net
greenhouses. One greenhouse was equipped with nutrient recycling system
while other without nutrient recycling as a control. The amount of
water and nutrient saved were assessed, and plant growth performance in
both greenhouses was compared.
Studies revealed that recirculation of nutrient solution could save
31.5% of total irrigation water. Among the measured major plant
essential elements, the potential saving of nitrogen, phosphorus,
potassium and calcium were 29.87%, 31.44%, 29.83% and 28.16%
respectively. The break-even of the additional cost of nutrient
recycling was less than five crop productions over a given area. The
total crop yield of the closed fertigated greenhouse was almost similar
to that of open fertigated greenhouse. Plant growth parameters (plant
height, stem diameter and leaf area index), average size and weight of
fruits, and fruit qualities (marketable fruits, moisture conte
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Heine, G., G. Tikum, W. J. Horst (2005)
Spatial sensitivity of tomato and bitter gourd roots to Pythium aphanidermatum infection as affected by silicon nutrition.
In: Proceedings of the XVth International Plant Nutrition Colloquium:
Plant nutrition for food security, human health and environmental
protection, Tsinghua University Press, Bejing, China, pp. 814-815.
ABSTRACT
The objectives of the present study were to quantitatively assess the infection success and the spread of Phythium aphanidermatum
along individual roots of the silicon (Si) excluder tomato and the Si
accumulator bitter gourd as affected by Si supply. For the application
of Si and zoospores to different root zones individual roots of intact
plants were mounted into PVC boxes with compartments sealed with
agarose. For the quantification of Pythium colonization of
roots a sensitive ELISA test was developed. Application of zoospores to
the apical and sub-apical root zones revealed that in tomato as well as
in bitter gourd the root tip is the most sensitive root section to Pythium infection leading to cessation of root growth. Application of Si both during pretreatment and during Pythium
treatment did not affect the infection levels in the root tip and
inhibition of root growth in either species. However, in bitter gourd
but not in tomato the basipetal spread of the fungus was inhibited.
Neither the treatment of the plants with Si only prior to or only
during Phythium infection reduced the spread of the fungus in the root. We conclude, that Si induces Pythium
defense mechanisms only in bitter gourd roots and this requires both
the presence and continued uptake of Si into the root symplasm.
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HEINE, G., TIKUM, G., HORST, W.J. (2005).
Silicon nutrition of tomato and bitter gourd with special emphasis on silicon distribution in root fractions.
J. Plant Nutr. Soil Sci. 168, 600-606.
SUMMARY
Two plant species, tomato (Lycopersicon esculentum Mill,) and bitter gourd (Momordica charantia),
were used for in-depth studies on the dynamics of silicon (Si) uptake
and translocation to the shoots and compartmentation of Si in the roots.
The experiments were conducted under controlled environmental
conditions in nutrient solutions which were partly amended with 1 mM Si
in the form of silicic acid. At harvest, xylem exudates were collected
and Si concentrations and biomass of roots and shoots were determined.
Mass flow of Si was calculated based on the Si concentration of the
nutrient solution and transpiration determined in a parallel
experiment. Plant roots were subjected to a fractionated Si analysis
allowing attributing Si to different root compartments.
Si concentrations in the roots compared to the shoots were higher in
tomato but lower in bitter gourd. A more ready translocation from the
roots to the shoots in bitter gourd, was in agreement with Si
concentrations in the xylem exudates which were higher than in the
external solution. In tomato the xylem-sap Si concentration was lower
than in the nutrient solution. Calculated Si mass flow to the root
exceeded Si uptake in tomato, which was consistent with the measured
accumulation of Si in the root water free-space (WFS). In contrast, Si
concentration in the root WFS was lower than in the nutrient solution
in bitter gourd reflecting the calculated Si depletion at the root
surface based on the comparison of Si mass flow and Si uptake.
Within the roots more than 80% of the total Si was located in the cell
wall and less than 10% in the cytoplasmic fractions in tomato. In
bitter gourd between 60% and 70% of the total root Si was attributed to
the cell-wall fraction whereas the proportion of the cytoplasmic
fraction reached more than 30%.
Our results clearly confirm that tomato belongs to the Si excluders and
bitter gourd to the Si accumulator plant species for which high Si
concentrations in the cytoplasmic root fraction appears to be
characteristic.
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Gregor Heine and Walter J. Horst (2004).
The role of silicon in the resistance of tomato against Pythium aphanidermatum.
Paper presented at the Deutscher Tropentag, Berlin, 2004.
ABSTRACT
Vegetable production in greenhouses is threatened by root rot caused by various Pythium species, among them P. aphanidermatum
which is a soil- borne plant pathogen with mainly tropical
distribution. It is well established that silicon nutrition can enhance
the resistance of cucumber against leaf diseases such as powdery mildew
but also against root rot caused by Pythium ultimum. Whether Si may have a comparable effect in tomato is subject of the present study.
In contrast to cucumber which belongs to the Si accumulator plants
characterized by a high Si uptake and translocation rate to the shoots,
our results clearly show that tomato can be classified as a Si excluder
discriminating Si from uptake. This is reflected in a lower Si
concentration in the xylem exudates and a characteristic accumulation
of Si in the root water free space and particularly in the cell walls
as revealed by a fractionation of Si in the roots.
In studies conducted under controlled conditions in nutrient solution and in a peat substrate two isolates of P. aphanidermatum
originating from vegetable production sites in Thailand showed to be
pathogenic for the tomato variety King Kong II. Methods for the
reliable production of zoospores and infection material on PDA plates
and in Maize brush/soil mixture were established.
When growing plants in nutrient solution, several plants died after
infection with zoospores but root length and shoot growth of the
surviving plants were not significantly different compared to the
non-inoculated control. Silicon nutrition had no influence on growth
parameters. When seeded into Pythium-infested
peat substrate most of the tomato plants died. However, transplanted
tomato plants (10 days old) generally survived the Pythium infection, but root growth and shoot weight were reduced.
Infection of the substrate with mycelium-infested maize brush/soil
mixture proved to cause a stronger infection than mixing the substrate
with homogenized PDA plates. Generally, the infection and the response
of the tomato plants were very variable. However, the experiments
clearly indicate that Si application to the substrate reduces plant
damage expressed in reduced root and shoot growth.
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