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PIXE studies of changes in mineral composition of plants infected with Phytophthora cinnamomi
Nuclear Instruments and Methods 181 (1981) 275-278 © North-Holland Publishing Company
PIXE STUDIES OF CHANGES IN MINERAL COMPOSITION OF PLANTS INFECTED WITH PHYTOPHTHORA CINNAMOMI M. Anwar CHAUDHRI Department of Medical Physics, Austin Hospital, Heidelberg, Australia 3084, and Department of Medicine at Austin, University of Melbourne, Australia C.S. PAPPER * School of Physics, University of Melbourne, Australia
and G. WESTE School of Botany, University of Melbourne, Australia
The mineral composition of susceptible and resistant plants from native forests infected with Phytophthora cinnamomi was compared between themselves and with the same species from disease-free areas. Root and shoot samples from different plants were carefully ashed, compressed into pellets and analysed with the thick target PIXE technique. A number of elements, Ca, Ti, Mn, Fe, Ni, Cu, Zn, Rb, Sr, Zr, Sn and Pb, were detected. Many of the elements showed variations, sometimes large, between the composition of susceptible and resistant plants, and between the same species from infected and disease-free forests.
1. Introduction
affected plants. PIXE is particularly suited to this type o f comparative study in similar matrices. It provides simultaneous information on most elements heavier than Na with good detection sensitivity. It is further simplified in the present case as the relative comparison o f the elemental concentrations in diseased and unaffected plants, or in plants from diseased and healthy areas, do not require absolute values. Moreover, as the samples being compared have similar matrices, there is no need for any matrix correction. A recent study has demonstrated that infected roots o f Eucalyptus obliqua were deficient in Fe and Ti compared with disease-free roots [2]. This project compares the mineral content o f susceptible and resistant plants when grown in diseased native forests with the same species grown in disease-free areas. Moreover, it also compares the compositions o f the two species grown in diseased and disease-free forests.
It has been shown that Phythophthora cinnamorni causes severe disease in the dry shrubby forests of the Brisbane Ranges, Victoria [1]. Most of the species comprising the woody flora are susceptible to the pathogen. While the primary s y m p t o m is root rot, the first obvious indication o f infection by the fungus is the yellowing, or chlorosis, of the foliage and of the plant apices, which results in die back. Leaves turn yellow, then brown and finally the host dies, usuaUy with the leaves attached. There is no recovery. "Little leaf" is also a characteristic s y m p t o m o f the disease for some species. Chlorosis m a y indicate interference with mineral uptake by the root and also with the mobility o f minerals within plant tissue. In order to study these effects of Phytophthora cinnamomi, it was decided to examine the mineral composition o f diseased plants and compare it with that of unaffected specimens grown under the same conditions, The technique o f thick-target PIXE was chosen to compare the mineral composition o f diseased and un-
2. Experimental methods Plants were sampled from native dry sclerophyll forest in Wilsons Promontory, Species selected included 10 plants of ceratophyllus which is susceptible and 10
* Present address: Pilkington-A.C.I., Special Products Division, Dandenong, Australia 3175. 275
shrubby Victoria. Isopogon plants of
X. ANALYSIS OF BIOLOGICAL SAMPLES
276
M.A. Chaudhri et al. / Changes in mineral composition
Gahnia redula which is resistant in that it invades and
flourishes in diseased forests and is free from any pathogen. Five plants of each species were sampled at random from the diseased forest and five from the nearby unaffected forest with similar soil, vegetation and topography. Each plant was removed intact with surrounding soil and placed in a separate plastic bag. The presence of the pathogen in diseased forest samples and its absence from unaffected samples was determined by lupin baiting of the soil [3], and subsequent isolation onto antibiotic agar [4]. The contents of each sample bag were separated into root and shoot, dried at I05°C for three days and ground finely. Stem and root samples were ashed at 400°C for 48 h, resulting in a fine grey white powder. The powder samples were compressed into pellets of 2 mm thickness in aluminium holders, using a hydraulic press. A thin layer of carbon was evaporated over all the targets to eliminate charging during proton bombardment [5]. All the samples were mounted in the alumininm target ladder of the scattering chamber at 45 ° with the incident beam. The chamber assembly was insulated, and was used for monitoring the beam current. The target ladder could be manipulated externaUy without breaking the vacuum, and any one of the targets placed in the beam. Samples were bombarded with 2 MeV protons at an intensity of 1 5 20 nA. The resulting X-rays passed through a 25 #m Kapton window at 90 ° in the chamber and were measured with an Ortec Low Energy Photon detector, conventional electronics and a PDP-11 computeranalyser system. Two 0.25 mm thick Hostaphan filters were placed in front of the detector to reduce the intensity of X-rays from lighter elements which have large concentrations in plants. The count rate in the detector was kept to around 1000 cps, in order to achieve the best possible resolution from the detector and to avoid any pile-up effects and excessive dead time. No standards were used in this experiment as the elemental concentrations in different samples were not required in absolute units for this comparative study.
~C
S
-
r
~
T¸
-
1 .u
z
J
Sr
10 3
/G2 10' I
Zn AsL ~ i
200
a
j/~
I
300
400 508 600 70{; 3HANNt[ NUMBER
~ 800
900
Fig. 1. PIXE spectrum t¥om a thick compressed target of the ashed roots from a resistant plant grown in an infected forest.
sponding to different elements in the samples are identified and labelled accordingly. The background subtraction, peak area integration, extraction of overlapping peaks and estimation of errors were carried out using the PDP-11 computer. The areas of various peaks are proportional to the concentrations of tire corresponding elements, and can be used for comparative study without having to convert them into absolute units. This is because the matrices of all the samples investigated are very similar, and therefore, no matrix absorption corrections are required. In this way the concentrations of all the elements observed: Ca, Ti, Mn, Fe, Ni, Cu, Zn, Rb, Sr, Zr, Sn and Pb, in different plant samples were calculated. For a comparison of the mineral composi-
]Cs ~,tn Fe 0o
1
AIIA o
103
h /~ /If
~ ~m!
I
~,
Pb I ~ Sr
Zr
1
Rb
3. Results and discussion
P1XE spectra from the roots of the resistant plant, grown in infected and disease-free forests, are shown in figs. 1 and 2 respectively. A number of peaks corre-
2019
300
408
500
600
70C
800
9GO
C'flnNNELNUHSEP Fig. 2. PIXE spectrum from a thick compressed target of the ashed roots from a resistant plant grown in a disease-free forest.
M.A. Chaudhri et al. / Changes in mineral composition
277
Table 1 P1XE results on the comparison of mineral composition for shoots.
Ca Ti Mn Fe Ni Cu Zn Rb Sr Pb
Y (Tolerant)/Y (Susceptible)
Y (Diseased)/Y (Healthy)
Diseased area
Healthy area
Susceptible
Tolerant
0.310 1.419 0.207 1.106
+- 0.011 ± 0.180 ,+ 0.023 ,+ 0.025
,+ 0.085 ,+ 0.098 ,+ 0.064 + 0.045
+- 0.073 +- 0.292 ,+ 0.153 -+ 0.023 -+ 0.490
0.860 1.597 0.754 1.716 0.570 1.459 1.737 1.372 0,895 0.804
1.410 0.893 0.543 1.494
0.699 1.458 0.300 0.498 1.082
0.220 1.900 0.315 1.164 0.408 1.721 0.718 1.039 0.431 0,681
0.800 1.873 0.369 1.218 1,972
+_0.102 -+ 0.619 ,+ 0.192 ,+ 0.090 + 0.922
+ 0.011 + 0.239 + 0.014 _+0.029 ± 0.179 ,+ 0.157 + 0.239 ,+ 0.272 ,+ 0.020 + 0.271
t i o n s o f d i f f e r e n t species, and o f t h e same p l a n t
-+ 0.043 ,+ 0.208 -+ 0.021 + 0.038 + 0.187 ± 0.128 _+0.339 ± 0.320 ± 0.031 + 0.279
In the tables a ratio o f u n i t y w o u l d m e a n t h a t t h e r e is n o d i f f e r e n c e b e t w e e n the t w o q u a n t i t i e s
species g r o w n in i n f e c t e d and disease-free areas, the ratios o f the respective e l e m e n t a l c o n c e n t r a t i o n s (Y:
c o m p a r e d . H o w e v e r , as can be seen f r o m b o t h the tables, m o s t o f t h e ratios are d i f f e r e n t f r o m u n i t y .
t h e X-ray yield) w e r e calculated. These results are s u m m a r i z e d in tables 1 and 2, w h i c h r e p r e s e n t the
This m e a n s t h a t t h e c o n c e n t r a t i o n s o f a n u m b e r o f
results for s h o o t s and r o o t s respectively. In these tables the ratios o f the mineral c o m p o s i t i o n s o f the
elements
vary
between
susceptible
and
resistant
r e s i s t a n t / t o l e r a n t and susceptible plants are s h o w n for b o t h d i s e a s e d / i n f e c t e d and h e a l t h y / d i s e a s e - f r e e areas.
plants for b o t h the i n f e c t e d and disease-free areas. The same is true w h e n the m i n e r a l c o m p o s i t i o n o f t h e same species is c o m p a r e d b e t w e e n the i n f e c t e d and
M o r e o v e r , the tables include c o m p a r i s o n s (ratios) o f t h e mineral c o m p o s i t i o n o f the same species, g r o w n in i n f e c t e d a n d disease-free areas. This has b e e n d o n e
disease-free forests. These variations in the e l e m e n t a l c o n c e n t r a t i o n s are a bit t o o m a n y t o be described here individually. These can, h o w e v e r , be easily seen
for susceptible a n d t o l e r a n t / r e s i s t a n t plants separately. The errors s h o w n in the tables are e x p e r i m e n t a l , a n d are mainly due to the c o u n t i n g statistics, e x t r a c t i o n
f r o m t h e tables w h i c h are s e l f - e x p l a n a t o r y . The biological significance o f t h e d i f f e r e n c e s in t h e mineral c o m p o s i t i o n s o f the susceptible a n d resistant species, and b e t w e e n the same species f r o m i n f e c t e d
o f the o v e r l a p p i n g peaks and b a c k g r o u n d s u b t r a c t i o n .
Table 2 PIXE results on the comparison of mineral composition for shoots.
Ca Ti Mn Fe Ni Cu Zn Rb Sr Zr Sn Pb
Y (Tolerant)/Y (Susceptible)
Y (Diseased)/Y (Healthy)
Diseased area
Healthy area
Susceptible
Tolerant
0.296 0.682 0.514 1.590
± 0.015 + 0.025 + 0.153 + 0.025
0.156 5.543 0.426 2.663
_+0.008 -+ 0.448 -+ 0.076 ,+ 0.053
1.513 0.669 0.677 1.090
-+ 0.076 + 0.024 ,+ 0.225 +- 0.013
1.698 3.796 0.338 0.553
+ 0.049 ,+ 0.616 +- 0.113 ,+ 0.020
0.734 1.274 0.991 0.220
+ 0.029 -+ 0.178 + 0.233 -+ 0.015
0.797 5.439 0.562 1.825 2.675 1.342 0.691 1.401 1.223
3.102 2.060 0.477 3.074 1.385
+ 0.107 + 0.242 -+ 0.158 -+ 0.218 + 0.205
0.709 ,+ 0.119
0.335 -+ 0.090
_+0.040 +_0.443 + 0.051 -+ 0.041 ,+ 0.509 + 0.047 + 0.124 ,+ 0.338 ,+ 0.037
2.155 ,+ 0.586 0.955 +- 0.149
2.012 ÷ 0.560
X. ANALYSIS OF BIOLOGICAL SAMPLES
278
M.A. Chaudhri et al. / Changes in mineral composition
and disease-free forests, is outside the scope of this paper and will be published elsewhere [6].
References [1] G. Weste and P. Taylor, Aust. J. Bot. 19 (1971) 281.
[2] M.A. Chaudhri, M.M. Lee, J.L. Rouse and G. Weste, Int. J. Appl. Rad. Isotopes 29 (1978) 585. [3] K. Chee and F.J. Newhook, N.Z.J. Agric. Res. 8 (1965) 88. [4] J.W. Eckert and P.H. Taso, Phytopath, 52 (1962) 771. [5] C.S. Papper, M.A. Chaudhri and J.L. Rouse, Nucl. Instr. and Meth. 154 (1978) 219. [6] G. Weste and M.A. Chaudhri, to be published.
PIXE STUDIES OF CHANGES IN MINERAL COMPOSITION OF PLANTS INFECTED WITH PHYTOPHTHORA CINNAMOMI M. Anwar CHAUDHRI Department of Medical Physics, Austin Hospital, Heidelberg, Australia 3084, and Department of Medicine at Austin, University of Melbourne, Australia C.S. PAPPER * School of Physics, University of Melbourne, Australia
and G. WESTE School of Botany, University of Melbourne, Australia
The mineral composition of susceptible and resistant plants from native forests infected with Phytophthora cinnamomi was compared between themselves and with the same species from disease-free areas. Root and shoot samples from different plants were carefully ashed, compressed into pellets and analysed with the thick target PIXE technique. A number of elements, Ca, Ti, Mn, Fe, Ni, Cu, Zn, Rb, Sr, Zr, Sn and Pb, were detected. Many of the elements showed variations, sometimes large, between the composition of susceptible and resistant plants, and between the same species from infected and disease-free forests.
1. Introduction
affected plants. PIXE is particularly suited to this type o f comparative study in similar matrices. It provides simultaneous information on most elements heavier than Na with good detection sensitivity. It is further simplified in the present case as the relative comparison o f the elemental concentrations in diseased and unaffected plants, or in plants from diseased and healthy areas, do not require absolute values. Moreover, as the samples being compared have similar matrices, there is no need for any matrix correction. A recent study has demonstrated that infected roots o f Eucalyptus obliqua were deficient in Fe and Ti compared with disease-free roots [2]. This project compares the mineral content o f susceptible and resistant plants when grown in diseased native forests with the same species grown in disease-free areas. Moreover, it also compares the compositions o f the two species grown in diseased and disease-free forests.
It has been shown that Phythophthora cinnamorni causes severe disease in the dry shrubby forests of the Brisbane Ranges, Victoria [1]. Most of the species comprising the woody flora are susceptible to the pathogen. While the primary s y m p t o m is root rot, the first obvious indication o f infection by the fungus is the yellowing, or chlorosis, of the foliage and of the plant apices, which results in die back. Leaves turn yellow, then brown and finally the host dies, usuaUy with the leaves attached. There is no recovery. "Little leaf" is also a characteristic s y m p t o m o f the disease for some species. Chlorosis m a y indicate interference with mineral uptake by the root and also with the mobility o f minerals within plant tissue. In order to study these effects of Phytophthora cinnamomi, it was decided to examine the mineral composition o f diseased plants and compare it with that of unaffected specimens grown under the same conditions, The technique o f thick-target PIXE was chosen to compare the mineral composition o f diseased and un-
2. Experimental methods Plants were sampled from native dry sclerophyll forest in Wilsons Promontory, Species selected included 10 plants of ceratophyllus which is susceptible and 10
* Present address: Pilkington-A.C.I., Special Products Division, Dandenong, Australia 3175. 275
shrubby Victoria. Isopogon plants of
X. ANALYSIS OF BIOLOGICAL SAMPLES
276
M.A. Chaudhri et al. / Changes in mineral composition
Gahnia redula which is resistant in that it invades and
flourishes in diseased forests and is free from any pathogen. Five plants of each species were sampled at random from the diseased forest and five from the nearby unaffected forest with similar soil, vegetation and topography. Each plant was removed intact with surrounding soil and placed in a separate plastic bag. The presence of the pathogen in diseased forest samples and its absence from unaffected samples was determined by lupin baiting of the soil [3], and subsequent isolation onto antibiotic agar [4]. The contents of each sample bag were separated into root and shoot, dried at I05°C for three days and ground finely. Stem and root samples were ashed at 400°C for 48 h, resulting in a fine grey white powder. The powder samples were compressed into pellets of 2 mm thickness in aluminium holders, using a hydraulic press. A thin layer of carbon was evaporated over all the targets to eliminate charging during proton bombardment [5]. All the samples were mounted in the alumininm target ladder of the scattering chamber at 45 ° with the incident beam. The chamber assembly was insulated, and was used for monitoring the beam current. The target ladder could be manipulated externaUy without breaking the vacuum, and any one of the targets placed in the beam. Samples were bombarded with 2 MeV protons at an intensity of 1 5 20 nA. The resulting X-rays passed through a 25 #m Kapton window at 90 ° in the chamber and were measured with an Ortec Low Energy Photon detector, conventional electronics and a PDP-11 computeranalyser system. Two 0.25 mm thick Hostaphan filters were placed in front of the detector to reduce the intensity of X-rays from lighter elements which have large concentrations in plants. The count rate in the detector was kept to around 1000 cps, in order to achieve the best possible resolution from the detector and to avoid any pile-up effects and excessive dead time. No standards were used in this experiment as the elemental concentrations in different samples were not required in absolute units for this comparative study.
~C
S
-
r
~
T¸
-
1 .u
z
J
Sr
10 3
/G2 10' I
Zn AsL ~ i
200
a
j/~
I
300
400 508 600 70{; 3HANNt[ NUMBER
~ 800
900
Fig. 1. PIXE spectrum t¥om a thick compressed target of the ashed roots from a resistant plant grown in an infected forest.
sponding to different elements in the samples are identified and labelled accordingly. The background subtraction, peak area integration, extraction of overlapping peaks and estimation of errors were carried out using the PDP-11 computer. The areas of various peaks are proportional to the concentrations of tire corresponding elements, and can be used for comparative study without having to convert them into absolute units. This is because the matrices of all the samples investigated are very similar, and therefore, no matrix absorption corrections are required. In this way the concentrations of all the elements observed: Ca, Ti, Mn, Fe, Ni, Cu, Zn, Rb, Sr, Zr, Sn and Pb, in different plant samples were calculated. For a comparison of the mineral composi-
]Cs ~,tn Fe 0o
1
AIIA o
103
h /~ /If
~ ~m!
I
~,
Pb I ~ Sr
Zr
1
Rb
3. Results and discussion
P1XE spectra from the roots of the resistant plant, grown in infected and disease-free forests, are shown in figs. 1 and 2 respectively. A number of peaks corre-
2019
300
408
500
600
70C
800
9GO
C'flnNNELNUHSEP Fig. 2. PIXE spectrum from a thick compressed target of the ashed roots from a resistant plant grown in a disease-free forest.
M.A. Chaudhri et al. / Changes in mineral composition
277
Table 1 P1XE results on the comparison of mineral composition for shoots.
Ca Ti Mn Fe Ni Cu Zn Rb Sr Pb
Y (Tolerant)/Y (Susceptible)
Y (Diseased)/Y (Healthy)
Diseased area
Healthy area
Susceptible
Tolerant
0.310 1.419 0.207 1.106
+- 0.011 ± 0.180 ,+ 0.023 ,+ 0.025
,+ 0.085 ,+ 0.098 ,+ 0.064 + 0.045
+- 0.073 +- 0.292 ,+ 0.153 -+ 0.023 -+ 0.490
0.860 1.597 0.754 1.716 0.570 1.459 1.737 1.372 0,895 0.804
1.410 0.893 0.543 1.494
0.699 1.458 0.300 0.498 1.082
0.220 1.900 0.315 1.164 0.408 1.721 0.718 1.039 0.431 0,681
0.800 1.873 0.369 1.218 1,972
+_0.102 -+ 0.619 ,+ 0.192 ,+ 0.090 + 0.922
+ 0.011 + 0.239 + 0.014 _+0.029 ± 0.179 ,+ 0.157 + 0.239 ,+ 0.272 ,+ 0.020 + 0.271
t i o n s o f d i f f e r e n t species, and o f t h e same p l a n t
-+ 0.043 ,+ 0.208 -+ 0.021 + 0.038 + 0.187 ± 0.128 _+0.339 ± 0.320 ± 0.031 + 0.279
In the tables a ratio o f u n i t y w o u l d m e a n t h a t t h e r e is n o d i f f e r e n c e b e t w e e n the t w o q u a n t i t i e s
species g r o w n in i n f e c t e d and disease-free areas, the ratios o f the respective e l e m e n t a l c o n c e n t r a t i o n s (Y:
c o m p a r e d . H o w e v e r , as can be seen f r o m b o t h the tables, m o s t o f t h e ratios are d i f f e r e n t f r o m u n i t y .
t h e X-ray yield) w e r e calculated. These results are s u m m a r i z e d in tables 1 and 2, w h i c h r e p r e s e n t the
This m e a n s t h a t t h e c o n c e n t r a t i o n s o f a n u m b e r o f
results for s h o o t s and r o o t s respectively. In these tables the ratios o f the mineral c o m p o s i t i o n s o f the
elements
vary
between
susceptible
and
resistant
r e s i s t a n t / t o l e r a n t and susceptible plants are s h o w n for b o t h d i s e a s e d / i n f e c t e d and h e a l t h y / d i s e a s e - f r e e areas.
plants for b o t h the i n f e c t e d and disease-free areas. The same is true w h e n the m i n e r a l c o m p o s i t i o n o f t h e same species is c o m p a r e d b e t w e e n the i n f e c t e d and
M o r e o v e r , the tables include c o m p a r i s o n s (ratios) o f t h e mineral c o m p o s i t i o n o f the same species, g r o w n in i n f e c t e d a n d disease-free areas. This has b e e n d o n e
disease-free forests. These variations in the e l e m e n t a l c o n c e n t r a t i o n s are a bit t o o m a n y t o be described here individually. These can, h o w e v e r , be easily seen
for susceptible a n d t o l e r a n t / r e s i s t a n t plants separately. The errors s h o w n in the tables are e x p e r i m e n t a l , a n d are mainly due to the c o u n t i n g statistics, e x t r a c t i o n
f r o m t h e tables w h i c h are s e l f - e x p l a n a t o r y . The biological significance o f t h e d i f f e r e n c e s in t h e mineral c o m p o s i t i o n s o f the susceptible a n d resistant species, and b e t w e e n the same species f r o m i n f e c t e d
o f the o v e r l a p p i n g peaks and b a c k g r o u n d s u b t r a c t i o n .
Table 2 PIXE results on the comparison of mineral composition for shoots.
Ca Ti Mn Fe Ni Cu Zn Rb Sr Zr Sn Pb
Y (Tolerant)/Y (Susceptible)
Y (Diseased)/Y (Healthy)
Diseased area
Healthy area
Susceptible
Tolerant
0.296 0.682 0.514 1.590
± 0.015 + 0.025 + 0.153 + 0.025
0.156 5.543 0.426 2.663
_+0.008 -+ 0.448 -+ 0.076 ,+ 0.053
1.513 0.669 0.677 1.090
-+ 0.076 + 0.024 ,+ 0.225 +- 0.013
1.698 3.796 0.338 0.553
+ 0.049 ,+ 0.616 +- 0.113 ,+ 0.020
0.734 1.274 0.991 0.220
+ 0.029 -+ 0.178 + 0.233 -+ 0.015
0.797 5.439 0.562 1.825 2.675 1.342 0.691 1.401 1.223
3.102 2.060 0.477 3.074 1.385
+ 0.107 + 0.242 -+ 0.158 -+ 0.218 + 0.205
0.709 ,+ 0.119
0.335 -+ 0.090
_+0.040 +_0.443 + 0.051 -+ 0.041 ,+ 0.509 + 0.047 + 0.124 ,+ 0.338 ,+ 0.037
2.155 ,+ 0.586 0.955 +- 0.149
2.012 ÷ 0.560
X. ANALYSIS OF BIOLOGICAL SAMPLES
278
M.A. Chaudhri et al. / Changes in mineral composition
and disease-free forests, is outside the scope of this paper and will be published elsewhere [6].
References [1] G. Weste and P. Taylor, Aust. J. Bot. 19 (1971) 281.
[2] M.A. Chaudhri, M.M. Lee, J.L. Rouse and G. Weste, Int. J. Appl. Rad. Isotopes 29 (1978) 585. [3] K. Chee and F.J. Newhook, N.Z.J. Agric. Res. 8 (1965) 88. [4] J.W. Eckert and P.H. Taso, Phytopath, 52 (1962) 771. [5] C.S. Papper, M.A. Chaudhri and J.L. Rouse, Nucl. Instr. and Meth. 154 (1978) 219. [6] G. Weste and M.A. Chaudhri, to be published.