Poacynum pictum from Apocynaceae family is widely
distributed in Xinjiang China. There are more than 6 ecotypes of the species
that can be distinguished from morphological and biological characteristics (Gao et al. 2015). P. pictum are
perennial herbaceous plants and their heights can be more than 4 m. Plants
leaves are used as a traditional Chinese medicine for the treatment of
hypertension, hepatitis, and depression (Zhang 2004; Xie
et al. 2012) and teas from the leaves of P. pictum have been used as a nutritional supplement in North American and East
Asian health food markets (Shi et al. 2011; Song and Zhou 2015). The fiber obtained
from the stem of the two species has become the interest in the textile
industry (Gong et al. 2017).
The leaves of Apocynaceae plants contains high
flavonoids content (Zhou et al. 2015) which has been considered to play a major
role in Chinese traditional medicines and tea (Nishibe
et al. 2001; Ma et
al. 2003), e.g., flavonoids isolated from
Apocynum venetum have significant anti-depressant activities for mice (Yan et al. 2015)
and inhibit the progress of
atherosclerosis in rats via the AMPK/mTOR pathway (Lü et al. 2017). Flavonoids have also been
noted to play a role in suppressing the growth of bacteria (Kang et al.
2014), such as Escherichia coli, and
highly decrease the ability of the bacteria to initiate an infection (Nguyen
et al. 2016; Shang et al. 2017). In tissues, the
substances may act as antioxidants and significantly contribute to scavenging
of free radicals produced via metabolic processes in plants. The leaves of Apocynaceae
plants contain metals such as Ca, Fe, Zn and Na which play important role in both plant
and animal metabolism (Yokozawa et al. 2002).
Rust caused by Melampsora apocyni
is the major disease affecting A. venetum and P. pictum, and all ecotypes have been found to be
affected by this species causing rust (Gao et al.
2017a). The disease has been reported in Russia (Tranzschel 1891),
Japan (Hiratsuka 1939), Kazakhstan (Nevodovskii
1956), China (Tai 1979), Bulgaria (Denchev 1995) and
Turkey (Kirbağ 2004). In Xinjiang China, we studied the rust on
leaves of wild and cultivated A. venetum plants from 2009 until now and this study is
ongoing. The rust occurrence could reach up to 100% in field conditions
cause the loss of leaves and also the death of plants in severe situations,
ultimately causing large economic
losses (Gao et al. 2017a).
Rust diseases have been reported to reduce the
crop yield include A. venetum
(Gao
et al. 2017a), which decrease the
crop protein and amino acid content in alfalfa and Vicia sativa (Nan 1986, 1990) and decrease the feed value of forage
plants. Reports have revealed that rust diseases could decrease plant amino acid
content by changing the metabolism of nitrogen in host plants (Nan 1986, 1990). Other reports found
that rust pathogens such as species
of Melampsora,
Phakopsora,
Puccinia
and Uromyces
can uptake amino acids in plants through intracellular haustoria,
thus decrease amino acid content (Struck
2015). Rust disease
has been reported to decrease the Ca and P
concentration in alfalfa plants and these metals also play role in the plant
disease defense system (Fones et al. 2010; Pinheiro et al. 2011). However, rust
infection has been reported to enhanced the
concentration of plant flavonoids (Miranda et al. 2007; Lu et
al. 2017).
Two ecotypes of P. pictum, one with red stems with medium
leaves referred to as ecotype RSM and another with green stems with fine leaves
that is referred to as ecotype GSF, are essential cultivated plants in Altay for
tea and fiber production (Gao et al.
2015). Rust has been found to be easily infect
these two ecotypes, and the average disease incidence could reach up to 70% in
cultivated fields. In this study, the effect of rust severity, based on a 6–step rating scheme and on series of
parameters related to the quality of P. pictum for tea production was analyzed. We hypothesized
that the infection of rust (M. apocyni)
will decrease the biomass, amino acid content and metal concentration and
increase the total flavonoids of these two ecotypes of P. pictum.
Materials and Methods
Experimental details and methods
Samples cultivation: The two
ecotypes of P. pictum,
Eco–RSM and Eco–GSF (Fig. 1), used in this study
were basically obtained as selections from local naturally-growing P. pictum plants.
They were cultivated in the Alakak Township in Altay Prefecture of the Xinjiang Uyghur
Autonomous Region, China (Alakak field, 47°42′N, 87°33′E, at an
altitude of 492 to 547 m, area 5.23
ha), using plants grown from seeds which were collected from locally growing
plants. The soil was a sandy
loam having pH of 7.2 to 7.5, and plant
rows were spaced 3 meters apart while within the rows
space was 1 m apart. Emergence of P. pictum stems began in the middle of April. P. pictum plants were irrigated by drip irrigation system after every 6 days and no
disease control measures were applied.
Rust disease survey
For each ecotype of P. pictum, four 20 × 20 m plots were used to assess the disease incidence of the
rust species. From each plot 200 leaves were sampled non–destructively
for each evaluation, and the disease incidence was recorded on leaves based on
the presence of uredinia. The rust severity
was recorded as a 6–step rating scheme by visually
calculation of the percentage of observed leaves that were covered by uredinia: 0 for no signs of infection; 1 for 0.1 to 5% of
leaf area covered with uredinia; 2 for 5.1 to 20%; 3
for 20.1 to 50%; 4 for 50.1 to 75%; and 5 for >75.1%. For each rust severity of the two P. pictum ecotypes
(Fig. 2), 100 leaves of similar
size from similar branches of the plants were sampled for dry weight, and total
flavonoids, amino acid and metal content were measured.
Total flavonoids extraction
According to this method, total flavonoids content (TFC) was calculated by
acid
hydrolysis method (Zhao et al. 2016) with little
changes. For each rust severity, 100 leaves were dried in an oven at 65°C for
24 h, the dry leaves were ground into fine powders and
mixed thoroughly. One-gram sample was precisely weighed and added to conical
flask containing 25 mL of 70% ethanol in a water bath with 65°C constant
temperature for 1h for reflux extraction. After cooling, 70% ethanol was added
to the 25 mL and was thoroughly mixed and filtered through quantitative filter
paper. One mL of the filtered solution was transferred to an evaporating dish,
1 g polyamide powder was added and the dish was kept in the water bath to
remove the ethanol. The sample was then transferred into a chromatographic
column, 20 mL of petroleum ether was added to remove impurities, the column was
then washed with 20 mL of methyl alcohol and the eluent was collected. This
eluent was allowed to dry. The residue was dissolved in methanol and
transferred into a 10 mL volumetric flask. Two mL of the solution was added to
10 mL volumetric flask, and then added 5 mL of 30% ethanol and 0.3 mL of 5%
sodium nitrite solution, and the solution was allowed to stand for 5 min. Then
0.3 mL of 10% nitric acid aluminum solution was added and the sample kept for
another 6 min. Finally, 2 mL of 1.0 mol L-1
sodium hydroxide solution was added. The absorbance was measured at 510 nm by
ultraviolet visible absorption spectrophotometer (UV-7502c, China).
Amino acid extraction
According to this method, amino acid extraction was measured by the Aluminium nitrate colorimetric method with minor
modification (Ksenofontov et al. 2017). The
fine powder of P. pictum
leaves as described in above method was used for amino acid extraction. For
each rust disease severity, 30 mg powdered samples were weighed and added
accurately to a hydrolysate tube in which 10 mL of 6 mol L-1 hydrochloric acid was added. The hydrolysate tube was evacuated by a vacuum pump and then
filled with nitrogen; this procedure was repeated 3 times and then the tube was
sealed with an alcohol blast burner. The tubes with samples were kept for 22 h
in a constant temperature oven at 110ºC to hydrolyze. The hydrolysate solutions were shifted to volumetric flasks and
made up to constant volume of 50 mL with deionized water. One mL of the
solution was added into a 5 mL of beaker and left to dry in glass desiccator.
The dried sample was dissolved with 1 mL of acetic acid buffer having 5.5 pH, and used to detect the amino acids presence by use
of an automatic amino acid analyzer (Hitachi 835, Japan).
Tryptophan
was determined by alkaline hydrolysis method. Powdered samples (30 mg) were
added accurately to a poly tetra fluoroethylene tube in which 3 mL of 5 mol L-1
sodium hydroxide was added. The tubes with samples were kept for 22 h in a
constant temperature oven at 110℃ to hydrolyze. The hydrolysate solutions
were shifted to 25 mL volumetric flasks and adjust pH to 7.6 after cooling,
made up to constant volume of 25 ml with deionized water. Finally, 1 ml of the
solution was shifted to 10 mL tube, made up to constant volume of 10 mL with 4
mol L-1 (pH 11) urea solution, and used to detect the Tryptophan
presence by use of a spectrofluorophotometer (Shimadzu RF-5000, Japan).
Metal evaluation
For mineral
content, 0.2 g of the dried samples were precisely weighed and dissolved in HNO3:HClO4:H2SO4
(8:1:1) mixture, and heated on a digestion furnace at 420°C in a fume hood.
After the digestion and subsequent cooling, the digested samples were added to
volumetric flasks and were diluted by the addition of ultrapure water to 100 mL. A flame atomic absorption spectrophotometer (Thermo ICE
3300, Germany) was used to measure metal elements (Zhang 2004).
Fig. 1: Eco RSM-red stem with medium
leaves (left) and Eco GSF-green stem with fine leaves (right) of P. pictum
Fig. 2: The rust severity
visually estimating the percentage of observed leaves that were covered by uredinia: 0 for no signs of infection; 1 for 0.1% to 5% of
leaf area covered with uredinia; 2 for 5.1% to 20%; 3
for 20.1% to 50%; 4 for 50.1% to 75%; and 5 for >75.1%. For each rust severity of the two P. pictum ecotypes
Statistical analysis
All data are presented as means and
standard errors of means for four
replicates. The significance of differences at a 5% level between averages was
determined by one-way ANOVA using Tukey's HSD test.
Results
Rust disease occurrence, biomass production
In the research sites, the rust was
firstly found in late July, and the two ecotypes of P. pictum had similar rust disease
incidence during the growth period. The disease incidence was kept under 20%
until the middle of August, and then there was a sharp increase from 20–60% in a week. Subsequently the
development of rust disease slowed down but reached up to 70% in two weeks, and
this persisted until leaves were lost and the growth period of the plants
ceased (Fig. 3). The infection of rust had no effect on leaf dry weight of P. pictum
plants of the GSF ecotype, however infection significantly decreased the dry
weight of leaves of the RSM ecotype at rust severity 4 (P < 0.05). There was no significantly difference between
rust-infected leaves with different rust severity (Fig. 4). Our hypothesis that
rust will decrease the biomass of the two ecotypes of P. pictum was partly supported (Table 1).
Total flavonoids concentration
The concentration of flavonoids in
healthy leaves of the two ecotypes is 2.4–2.8 g 100 g-1, which is
5.2–9.4% higher than rust–infected leaves. Compared with healthy leaves, the
infection of the rust pathogen M. apocyni only
significantly decreased flavonoids when the rust severity were 4 and 5 and the
flavonoids concentrations were 36.4 and 25.5% lower than healthy leaves,
respectively, for the RSM ecotype (Fig. 5). There was no significant difference
in flavonoid concentrations among leaves of all severities of leaves of the GSF
ecotype (Fig. 5 and Table 1). Rust disease had no significant effects on
flavonoids concentration in leaves of the GSF ecotype.
Table 1: ANOVA result of the
effects of rust disease caused by Melampsora apocyni on the listed variables to two ecotypes of Poacynum pictum
Variables |
Eco RSM -red stem with medium leaves |
Eco GSF-green stem with fine leaves |
||||
F values |
DF |
P values |
F values |
DF |
P values |
|
Leaf dry weight |
6.3478 |
5 |
0.0015 |
1.5728 |
4 |
0.2325 |
Disease incidence |
18.6747 |
3 |
0.0001 |
22.0470 |
3 |
0.0001 |
Flavonoids concentration |
3.9519 |
5 |
0.0136 |
0.4540 |
4 |
0.7681 |
Calcium concentration |
4.4805 |
5 |
0.0079 |
5.2512 |
4 |
0.0076 |
Copper concentration |
13.3846 |
5 |
0.0001 |
21.8334 |
4 |
0.0001 |
Zinc concentration |
7.4581 |
5 |
0.0006 |
0.8233 |
4 |
0.5304 |
Iron concentration |
3.7604 |
5 |
0.0166 |
2.3700 |
4 |
0.0991 |
Alanine |
24.1229 |
5 |
0.0001 |
11.3179 |
4 |
0.0002 |
Argnine |
34.1459 |
5 |
0.0001 |
16.0278 |
4 |
0.0001 |
Aspartic |
21.9949 |
5 |
0.0001 |
5.3065 |
4 |
0.0072 |
Cystine |
3.1365 |
5 |
0.0329 |
11.3432 |
4 |
0.0002 |
Glutamine |
22.0609 |
5 |
0.0001 |
11.9762 |
4 |
0.0001 |
Glycine |
26.2400 |
5 |
0.0001 |
9.6809 |
4 |
0.0004 |
Hlstidine |
26.3829 |
5 |
0.0001 |
11.2136 |
4 |
0.0002 |
Isoleucine |
24.0377 |
5 |
0.0001 |
9.7925 |
4 |
0.0004 |
Leucine |
28.0698 |
5 |
0.0001 |
13.4100 |
4 |
0.0001 |
Lysine |
21.4042 |
5 |
0.0001 |
11.5913 |
4 |
0.0002 |
Methionine |
2.6925 |
5 |
0.0550 |
6.1619 |
4 |
0.0039 |
Phenylalanine |
25.9132 |
5 |
0.0001 |
11.2994 |
4 |
0.0002 |
Proline |
24.5499 |
5 |
0.0001 |
11.1099 |
4 |
0.0002 |
Serine |
15.9173 |
5 |
0.0001 |
4.7319 |
4 |
0.0114 |
Threonine |
22.2670 |
5 |
0.0001 |
9.9547 |
4 |
0.0004 |
Tryptophan |
6.0371 |
5 |
0.0019 |
2.5727 |
4 |
0.0806 |
Tyrosine |
44.6389 |
5 |
0.0001 |
14.4998 |
4 |
0.0001 |
Valine |
24.2808 |
5 |
0.0001 |
10.0280 |
4 |
0.0004 |
Amino acid concentration
Compared with healthy leaves, amino
acid concentration was significantly affected (mainly decreased) by the rust
disease except for methionine and cystine in the RSM ecotype,
and tryptophan in the GSF ecotype. The decreases in the concentration of these
three amino acids happen under all rust disease severity (Table 1 and 2). P. pictum leaves with rust severity 4 and 5 had the lowest amino acid
concentration for the RSM ecotype. For the GSF ecotype the content of 10 and 16 of 18 amino acids was
reduced by rust disease at rust severity 3 and by the rust disease at
severity 4, respectively, rust severity 4 had significantly higher
amino acid content, 16 of 18 except Methionine and Trytophan,
than that of rust severity 1. When rust severity were 4 and 5, rust disease
significantly decreased the concentration of 15 amino acids in leaves of
the RSM ecotype, Except for Cystine, Methionine and Trypothan, which given the
same value under the different rust severity (Table 2).
The
correlations of rust disease severity and the amino acids depicts that amino
acid content is negatively correlated with rust disease severity for the two
ecotypes, except for Cystine, Methionine and Trytophan (Table 3). The amino acid response to rust disease
differed in the two ecotypes. With the GSF ecotype the content of Methionine
and Trytophan had no correlation with rust disease
severity. Glutamine is the most sensitive amino acid to rust disease, followed
by Leucine, while Trytophan
is the least sensitive amino acid to rust disease with both ecotypes of P. pictum (Table
2). Our hypothesis that rust will decrease the amino acid of the two ecotypes
of P. pictum
was upheld.
Table 2: Amino acid concentration of two ecotypes of Poacynum picttum under
different rust disease severity caused by Melampsora apocyni %
Rust disease severity of Ecotype red stem with
medium leave (Eco-RSM) |
Rust disease severity of Ecotype green stem with
fine leaves (Eco-GSF) |
||||||||||
0 |
1 |
2 |
3 |
4 |
5 |
0 |
1 |
2 |
3 |
4 |
|
Alanine |
1.22±0.05 a* |
1.24±0.05 a |
1.28±0.01 a |
1.11±0.06 a |
0.82±0.05 b |
0.77±0.04 b |
1.31±0.07 a |
1.23±0.05 ab |
1.04±0.04 bc |
1.08±0.06 ab |
0.84±0.05 c |
Argnine |
1.16±0.05 ab |
1.19±0.05 ab |
1.34±0.06 a |
1.01±0.05 b |
0.73±0.04 c |
0.68±0.03 c |
1.20±0.07 a |
1.10±0.05 ab |
0.93±0.01 bc |
0.92±0.03 bc |
0.73±0.05 c |
Aspartic |
1.76±0.08 a |
1.86±0.08 a |
1.97±0.05 a |
1.68±0.08 a |
1.24±0.07 b |
1.18±0.05 b |
1.87±0.10 a |
1.73±0.07 a |
1.55±0.07 ab |
1.60±0.10 ab |
1.32±0.10 b |
Cystine |
0.58±0.06 a |
0.55±0.02 a |
0.49±0.01a |
0.47±0.05 a |
0.39±0.03a |
0.46±0.03a |
0.62±0.02 a |
0.56±0.00ab |
0.51±0.01bc |
0.48±0.03bc |
0.46±0.02 c |
Glutamine |
2.41±0.08 a |
2.44±0.13 a |
2.39±0.01 a |
2.21±0.13a |
1.59±0.07 b |
1.53±0.07 b |
2.46±0.12 a |
2.26±0.09 ab |
1.97±0.03 bc |
1.97±0.08 bc |
1.65±0.10 c |
Glycine |
1.04±0.05 a |
1.08±0.05 ab |
1.15±0.02 ab |
0.95±0.04 b |
0.70±0.04 c |
0.66±0.03 c |
1.12±0.06 a |
1.04±0.05 ab |
0.90±0.02 bc |
0.93±0.05 abc |
0.74±0.04 c |
Hlstidine |
0.45±0.02 ab |
0.46±0.02 ab |
0.51±0.02 a |
0.41±0.02 b |
0.30±0.02 c |
0.28±0.02 c |
0.48±0.02 a |
0.44±0.02 ab |
0.38±0.01 bc |
0.40±0.02 ab |
0.30±0.02 c |
Isoleucine |
0.96±0.04 ab |
1.00±0.04 ab |
1.07±0.02 a |
0.89±0.04 b |
0.67±0.04 c |
0.64±0.03 c |
1.00±0.06 a |
0.96±0.04 ab |
0.82±0.02 bc |
0.83±0.03 abc |
0.69±0.04 c |
Leucine |
1.73±0.08 ab |
1.81±0.08 ab |
1.96±0.04 a |
1.56±0.08 b |
1.14±0.07 c |
1.07±0.06 c |
1.88±0.11 a |
1.75±0.08 ab |
1.47±0.03 bc |
1.48±0.05 bc |
1.19±0.07 c |
Lysine |
1.21±0.04 a |
1.23±0.05 a |
1.18±0.02 a |
1.09±0.06 a |
0.80±0.05 b |
0.76±0.04 b |
1.23±0.07 a |
1.15±0.05 ab |
1.01±0.01 bc |
1.02±0.03 bc |
0.83±0.04 c |
Methionine |
0.21±0.06 a |
0.08±0.00 a |
0.09±0.00 a |
0.08±0.01 a |
0.12±0.04 a |
0.10±0.02 a |
0.13±0.00 ab |
0.16±0.02 ab |
0.18±0.02 a |
0.13±0.01 b |
0.11±0.00 b |
Phenylalanine |
1.10±0.05 ab |
1.16±0.05 ab |
1.29±0.04 a |
1.03±0.05 b |
0.75±0.04 c |
0.70±0.04 c |
1.20±0.07 a |
1.13±0.05 ab |
0.97±0.02 bc |
0.98±0.04 bc |
0.80±0.04 c |
Proline |
0.92±0.03 a |
0.95±0.04 a |
1.00±0.02 a |
0.86±0.04 a |
0.63±0.03 b |
0.60±0.03 b |
0.98±0.05 a |
0.92±0.04 ab |
0.82±0.03 bc |
0.80±0.03 bc |
0.68±0.03 c |
Serine |
0.81±0.04 a |
0.83±0.03 a |
0.80±0.01 a |
0.74±0.04 a |
0.58±0.03 b |
0.55±0.03 b |
0.81±0.04 a |
0.83±0.03 a |
0.80±0.01 a |
0.74±0.04 a |
0.58±0.03 b |
Threonine |
0.91±0.04 a |
0.96±0.04 a |
0.96±0.00 a |
1.68±0.04 a |
0.63±0.03 b |
0.59±0.03 b |
0.98±0.05 a |
0.90±0.04 ab |
0.79±0.02 bc |
0.81±0.04 bc |
0.66±0.04 c |
Tryptophan |
0.13±0.01ab |
0.17±0.01 a |
0.14±0.01 a |
0.16±0.03 a |
0.07±0.01 b |
0.12±0.02 ab |
0.13±0.00 a |
0.12±0.00 a |
0.18±0.02 a |
0.16±0.02 a |
0.17±0.01a |
Tyrosine |
0.68±0.01 a |
0.65±0.02 a |
0.68±0.00 a |
0.53±0.03 b |
0.39±0.03 c |
0.37±0.01 c |
0.70±0.04 a |
0.65±0.03 ab |
0.53±0.01 bc |
0.54±0.02 bc |
0.42±0.03 c |
Valine |
1.21±0.04 a |
1.28±0.06 a |
1.35±0.03 a |
1.13±0.05 a |
0.84±0.05 b |
0.80±0.04 b |
1.28±0.07 a |
1.21±0.06 ab |
1.06±0.04 bc |
1.05±0.04 bc |
0.87±0.05 c |
Note:* Data marked by the same
lowercase letter in the same row do not differ significantly between rust
severity for the same ecotype of Pocynum pictum.
Metal concentration
Rust show different effects on
calcium (Ca), copper (Cu), iron (Fe) and zinc (Zn)
concentrations of the two ecotypes of P. pictum (Table 1). For the GSF ecotype, rust disease had
no effect on Fe and Zn concentration, while in case of severe rust infection
(severity 4) significantly (P < 0.05)
increased Ca and Cu concentrations. For the RSM
ecotype, when compared with healthy leaves, the concentration of Ca was significantly (P
< 0.05) decreased at rust severity 5, and with
Cu at rust severity 4 and 5. Healthy leaves had the same concentration of Zn
and Fe as with rust-infected leaves, but there was significant difference among
rust-infected leaves with varying rust severity. Rust severity 2 and 3 had
higher Zn concentrations than the other severities of rust infection. Rust
severities of 5 and 3 had the highest and lowest Fe concentration among
rust-infected leaves (Fig. 6). Our hypothesis that rust will decrease the metal
of the two ecotypes of P. pictum was partly upheld.
Discussion
Fig. 3: Rust disease incidence of two
ecotypes Eco RSM-red stem with medium leaves and
Eco–GSF green stem with fine leaves of P.
pictum in cultivated field. The same lowercase letters
up the bars means there is no significantly different by Turkey’s HSD at P < 0.05 for Eco RSM; The same uppercase
letters up the bars means there is no significantly different by Turkey’s HSD at P
< 0.05 for Eco GSF
Fig. 4: Leave dry weight of two ecotypes
Eco RSM-red stem with medium leaves and Eco
GSF-green stem with fine leaves of P. pictum in
cultivated field. The same lowercase letters up the bars means there is no
significantly different by Turkey’s HSD at P<0.05
for Eco RSM; the same uppercase letters up the bars means there is no
significantly different by Turkey’s HSD at P<0.05
for Eco GSF
Fig. 5: Flavonoids of two ecotypes Eco
RSM-red stem with medium leaves and Eco GSF-green stem with fine leaves of P. pictum in
cultivated field.
The same lowercase letters up the
bars
Means there is no significantly
different by Turkey’s HSD at P <
0.05 for Eco RSM; the same Uppercase letters up the
bars means there is no significantly different by Turkey’s HSD at P < 0.05 for Eco GSF
Our previous research showed that
infection by M. apocyni causes the leaves of A. venetum to turn yellow, wither and prematurely fall,
and results in significant yield loss in the field (Gao et al. 2015). In a greenhouse
experiment, infection by M. apocyni had a slight effect on photosynthesis of A. venetum
during early disease development, but drought stress was more damaging than for
non-inoculated plants in later disease development, leading to a great decrease
in the net photosynthetic rate This reduction, however, did not cause a
significantly decrease in the aboveground biomass of A. venetum plants between the
rust-infected and non-infected treatments (Gao et al. 2017b). The difference in leaf biomass of the two
ecotypes of P. pictum following rust infection
indicates the diversity of plant responses to this pathogen.
Table 3: Correlations of rust disease
severity caused by Melampsora apocyni and the listed variables to two ecotypes of Poacynum hendersonii
Variables |
Eco RSM-red stem with medium leaves |
Eco GSF-green stem with fine leaves |
||||
Coefficient |
Regression equation |
SE |
Coefficient |
Regression equation |
SE |
|
Leaf dry weight |
0.710** |
Y=0.721-0.038x |
0.067 |
0.453 |
Y=0.387-0.010x |
0.030 |
Flavonoids concentration |
0.621** |
Y=2.563-0.168x |
0.378 |
0.150 |
Y=2.177-0.044x |
0.435 |
Alanine |
0.820** |
Y=1.336-0.105x |
0.131 |
0.819** |
Y=1.319-0.109x |
0.114 |
Argnine |
0.790** |
Y=1.314-0.118x |
0.164 |
0.879** |
Y=1.202-0.113x |
0.092 |
Aspartic |
0.757** |
Y=1.974-0.144x |
0.222 |
0.722** |
Y=1.895-0.122x |
0.174 |
Cystine |
0.589 |
Y=0.566-0.030x |
0.074 |
0.852** |
Y=0.606-0.039x |
0.036 |
Glutamine |
0.836** |
Y=2.601-0.203x |
0.238 |
0.851** |
Y=2.444-0.192x |
0.177 |
Glycine |
0.796** |
Y=1.163-0.093x |
0.126 |
0.804** |
Y=1.118-0.086x |
0.095 |
Hlstidine |
0.772** |
Y=0.503-0.041x |
0.060 |
0.812** |
Y=0.477-0.039x |
0.042 |
Isoleucine |
0.775** |
Y=1.066-0.079x |
0.115 |
0.817** |
Y=1.010-0.076x |
0.080 |
Leucine |
0.790** |
Y=1.956-0.164x |
0.227 |
0.856** |
Y=1.884-0.165x |
0.148 |
Lysine |
0.850** |
Y=1.304-0.103x |
0.114 |
0.837** |
Y=1.234-0.094x |
0.092 |
Methionine |
0.332 |
Y=0.147-0.013x |
0.068 |
0.350 |
Y=0.158-0.008x |
0.333 |
Phenylalanine |
0.748** |
Y=1.257-0.100x |
0.159 |
0.837** |
Y=1.206-0.096x |
0.093 |
Proline |
0.794** |
Y=1.020-0.038x |
0.106 |
0.846** |
Y=0.982-0.071x |
0.067 |
Serine |
0.827** |
Y=0.087-0.061x |
0.074 |
0.613** |
Y=0.860-0.051x |
0.098 |
Threonine |
0.810** |
Y=1.006-0.076x |
0.098 |
0.814** |
Y=0.977-0.074x |
0.079 |
Tryptophan |
0.387 |
Y=0.154-0.009x |
0.040 |
0.446 |
Y=0.132+0.010x |
0.029 |
Tyrosine |
0.895** |
Y=0.726-0.071x |
0.063 |
0.861** |
Y=0.704-0.067x |
0.059 |
Valine |
0.776** |
Y=1.353-0.101x |
0.146 |
0.829** |
Y=1.289-0.098x |
0.098 |
Caconcentration |
0.555* |
Y=4.727-0.323x |
0.864 |
0.610* |
Y=8.563-0.910x |
1.763 |
Cu concentration |
0.816** |
Y=0.186-0.025x |
0.032 |
0.639* |
Y=0.132+0.070x |
0.126 |
Zn concentration |
0.384 |
Y=0.790-0.055x |
0.238 |
0.374 |
Y=2.142-0.180x |
0.667 |
Fe concentration |
0.235 |
Y=0.051+0.007x |
0.049 |
0.594* |
Y=0.166-0.022x |
0.045 |
Note:SE=standard error; *P < 0.05, **P < 0.01;
x=rust disease severity.
Fig. 6: Metal concentration of two
ecotypes Eco RSM-red stem with medium leaves and
Eco GSF-green stem with fine leaves
of P. pictum
in cultivated field. The same lowercase letters up the bars means there is no
significantly different by Turkey’s HSD at P<0.05
for Eco RSM; The same uppercase letters up the bars means there is no
significantly different by Turkey’s HSD at P<0.05
for Eco GSF
Several research studies have found
that the infection by pathogens increased plant flavonoids concentration in
plant tissues. An example of this was that Miranda et al. (2007) using the Populus 15.5K cDNA microarray, found that genes encoding enzymes required
for synthesis of the flavonoid proanthocyanidin were
up regulated dramatically. Phytochemical analysis confirmed that in late
infection, proanthocyanidin levels increased in
infected leaves. Lu et al. (2017)
also found that the amount of flavonoid compounds, especially
anthocyanin and catechin, were significantly
increased in rust-infected symptomatic tissue. The expression levels of
structural genes and MYB transcription factors related to flavonoid
biosynthesis were one to seven-fold higher in the
tissue infected by rust.
The present study indicates that the
two ecotypes of P. pictum
had different responses to rust infection, as rust had no effects on flavonoids
content of leaves of the GSF ecotype |but resulted in decreased flavonoids
concentration in the leaves of the RSM ecotype. This decrease with the RSM
ecotype is opposite to previous reports about the effects of rust disease on
plant flavonoids concentration. The dry weight and flavonoids of the two
ecotypes of P. pictum
had the same response to rust. The accumulation of carbohydrates in
rust-infected plants may be associated with the flavonoid biosynthesis pathway
(Wan et al. 2015) and
also, those carbohydrates which are a component of osmotic regulation during
pathogen infection, may contribute to the accumulation of flavonoids (Lu et al. 2017). This new
finding that rust decreases P. pictum flavonoids
is important supplementary knowledge of the effects of rust disease on plant
flavonoids as well as the evaluation of the resulting loss. It helps the understanding
of physiological effects of this rust species as found in our previous study in
a greenhouse that showed that the infection of rust changed activity of
peroxidase, polyphenol oxidase and phenylalanine ammonialyase in A.
venetum leaves (Gao et al. 2017b). Our hypothesis that rust will increase the total
flavonoids of the two ecotypes of P. pictum was not upheld.
Many reports depicted that rust disease decreases the amino acid content
of plants. For example, Nan found that the infection of rust (Uromyces onobrychidis) in Onobrychis viciaefolia, U. orobi
in Vicia sativa and U. striatus in alfalfa (Medicago sativa) and U. baeumlerianus in Melilotus albus, decreased total crude protein and
16 amino acids by more than 30% (Nan
1986, 1990). Rust fungi only can complete their life cycle on living
hosts where they grow through the leaf tissue by developing an extended network
of intercellular hyphae from which intracellular haustoria
are involved in suppressing host defense responses and acquiring nutrients.
Three amino acid transporter genes of the rust fungi, Uf–AAT1, Uf–AAT2 and Uf–AAT3, are closely
related with intracellular haustoria of rust fungi.
AAT1 and AAT3 are expressed very early during rust development and are strongly
up-regulated in haustoria (Struck et al.
2002, 2004) while AAT2 was shown to be strictly haustorium
specific (Hahn and Mendgen
1997). The decrease in amino acid content with severe infection of rust
disease in the two ecotypes of P. pictum may partly be due to the regulation of the
expression of these three transporter genes by the rust pathogen, as well as
the consumption, metabolism or the storage of amino acid by the pathogens (Hahn and Mendgen 1997;
Struck et al. 2002, 2004). There is also research found that pathogens
such as Pseudopeziza medicaginis
in alfalfa decrease the content of amino acids in host plants (Morgan and Parbery
1980). Even a low level of infection by Drechslera
siccans or Rhynchosporium
spp. significantly reduced in vitro dry matter digestibility, and water-soluble
carbohydrate and the total amino acid content of Italian ryegrass (Lolium multiflorum) and tall fescue (Festuca arundinacea),
and the decreases are correlated with the nitrogen metabolism and
transportation in the plant–pathogen
system.
Essential and non–essential heavy metals, such as Cu
and Zn, are quantified
in selected medicinal plants, including A.
venetum and P.
pictum, which are extensively used in the
preparation of herbal medicines for heart disease and tonics for general human
health. Rust disease has been shown decreased the content of Ca and P in alfalfa (Nan 1986), but the mechanism is not
clear. The defensive properties of metals against diseases of plants has
received much attention and support (Fones et al.
2010; Fones and Preston 2013), an example of
which is the supply of Ca and K reduces soybean rust
(Phakopsora pachyrhizi)
area under the disease progress curve (PUDPCS) (Pinheiro et al. 2011). The
accumulation of metals in plants is correlated with the activity of metal
transporters, e.g., repeated duplication of the gene encoding the P–type ATPase, HMA4, which is
responsible for xylem loading of Zn and cadmium (Cd) (Hanikenne et al. 2008).
Conclusion
Rust disease was caused by Melampsora apocyni
widely occur in field of cultivated P. pictum, and the disease incidence could reach up to 70%
for the two ecotypes. The severe occurrence of rust disease led to the decrease
of leaf biomass and flavonoids in the RSM ecotype, reduced the amino acid
content of the two ecotypes, and increased or decreased Ca
and Cu for the GSF and RSM ecotypes, respectively. These changes due to rust
infection decreased the value of P. pictum for tea and Chinese traditional medicine
production. Control methods for this rust disease are urgently required in this
region.
Acknowledgements
This research was financially
supported by Key Project of Science and Technology Department of Xinjiang
Autonomous Region, China (2016E02015, 2016A03006).
Author Contributions
Tingyu Duan and Peng Gao designed the experiment and analyzed the data; Yanru Lan performed the experiments;
Tingyu Duan wrote the
manuscript.
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