Journal of Agricultural and Food Chemistry
Article
compounds) and a training set (23 compounds). The EC50 values of
the compounds were converted into pEC50 and uploaded to the
server. The CoMFA analysis was performed using the partial least-
squares (PLS) regression approach. The cross-validation correlation
coefficient (q2), non-cross-validated correlation coefficient (r2), and
predictive correlation coefficient (r2pred), which were used to evaluate
the model, were obtained from the Web server. The force field model
was visualized using PyMol software (version 1.7.0.0), and the
compound with the highest antibacterial activity in the training set
was used as the template molecule.
Determination of Chlorophyll Content. The total chlorophyll
(Ct), chlorophyll a (Ca), and chlorophyll b (Cb) contents were tested
following a method previously reported.26
Proteomics Analysis. Plant Material and Sampling. Rice plants
grown under greenhouse conditions for 30 days were sprayed with
compound 33 (200 mg/L) until wet and infected with Xoc after 24 h.
The rice samples then were collected, snap-frozen, and stored at −80
°C.
Extraction and Identification of Total Rice Protein. The method
of protein extraction and identification was described in our previous
work.27
Morphological Change. Cell morphological changes after
treatment with compound 33 were observed by scanning electron
microscopy.23 After the bacteria were washed three times, phosphate-
buffered saline (PBS) was added to obtain a total volume of 1 mL,
and compound 33 dissolved in DMSO was added at concentrations of
2, 5, 25, and 200 mg/L. The same volume of DMSO in 1 mL of PBS
served as a negative control. After being shaken at 28 °C for 12 h, the
mixture was washed three times with PBS, and 2.5% glutaraldehyde
was added to fix the cells for 10 h. PBS was added to wash off the
glutaraldehyde, and then the cells were dehydrated with 10%, 30%,
50%, 70%, 90%, and 100% ethanol in turn. The morphological
changes were visualized using a scanning electron microscope after
the samples were freeze-dried for 4.5 h and coated with gold.
In Vivo Antibacterial Activity Test. The curative and protective
activities against BLB were evaluated according to the leaf-cutting
method.24 Commercial agents BT and TC were formulated into a
solution of the same concentration and served as positive controls,
and the same concentration solution without compounds served as a
negative control. Fengyouxiangzhan rice seeds were sown and grown
under greenhouse conditions for 5 weeks. The curative activity was
evaluated as follows. Xoo was inoculated in the NB medium and
cultivated until the logarithmic growth phase. The rice leaves were cut
2−3 cm from the leaf tips with sterile surgical scissors, and the rice
wounds were immersed in the NB medium with Xoo for 15 s. One day
after inoculation, a solution containing 200 mg/L of compound 33
was sprayed evenly on the rice leaves until droplets fell, and the
positive and negative controls were treated in the same way. The
protective activity was evaluated similarly. The solution containing the
compound was first evenly sprayed onto the rice leaves until droplets
fell, and Xoo was inoculated onto the rice the next day. The positive
and negative controls were treated in the same way. After inoculation
for 14 days, the antibacterial activity could be calculated from the
disease index.12
The curative and protective activities against BLS were tested by a
similar method but using a different inoculation method that was
reported in the previous literature with slight modifications.25 To
evaluate the curative activity, Xoc cultured to the logarithmic growth
phase was used to spot-infiltrate rice leaves with needleless syringes.
After 1 day, 0.1% Tween-20 sterile water containing compound 33 (at
a concentration of 200 mg/L) was sprayed evenly onto the rice leaves
until droplets fell. The positive and negative controls were treated in
the same way. For protective activity, the bacteria were inoculated 1
day after spraying compound 33. The lesion length on the rice was
surveyed 14 days later. The equation below illustrates the methods to
calculate the curative and protective activities against Xoc. In this
equation, L indicates the lesion length of the negative control group,
and T indicates the lesion length of the treatment group.
Bioinformatics Analysis. Gene ontology (GO) annotations were
independent categories: molecular functions (MFs), biological
processes (BPs), and cellular components (CCs). All differentially
expressed proteins (DEPs, fold change > 1.5, p-value < 0.05) were
mapped in the GO database, and the number of proteins mapped to
each GO term was computed. Meanwhile, Kyoto Encyclopedia of
Genes and Genomes (KEGG) annotations were performed using the
RESULTS AND DISCUSSION
■
Chemistry. The different substitutes of 4-amino-1,2,4-
triazole-3-thiol (A1−A7) were prepared by 2-(4-
chlorophenoxy)acetohydrazide or carbonothioic dihydrazide.
The intermediates A1−A7 were refluxed with carbon disulfide
in a KOH solution of methanol to give 1,2,4-triazolo[3,4-
b][1,3,4]thiadiazole-6-thiol. Subsequently, target compounds
1−33 were prepared by thioetherification and oxidation
reactions. The synthesis methods of the intermediates C1−
C33 and the data for all of the target compounds (melting
point, yield, 1H NMR, 13C NMR, and HRMS) are presented in
(CCDC 2041989, Figure S1) was obtained by a slow
evaporation of the DCM/EtOH solution and confirmed by
X-ray single-crystal diffraction analysis.
In Vitro Antibacterial Activity. The turbidimetric
method was used to test the biological activities of target
compounds 1−33. As shown in Table 1, target compounds 1−
33 showed good in vitro antibacterial activities against Xoc and
Xoo. The EC50 values of compounds 8, 9, 11, 15, 19, and 28
against Xoo were 0.97, 0.84, 0.74, 0.82, 0.88, and 0.88 mg/L,
respectively, which were better than those of TC (90.43 mg/L)
and BT (68.37 mg/L). Moreover, the EC50 values of
compounds 8, 9, 11, and 12 against Xoc were 1.58, 1.72,
1.74, and 1.71 mg/L, respectively, which were better than
those of TC (97.93 mg/L) and BT (75.59 mg/L).
3D-QSAR Analysis. We built CoMFA models on the basis
of the EC50 values of compounds 1−31 using the Cloud 3D-
values of the anti-Xoo
pred
CoMFA model, which were used to evaluate the predictive
ability of the model (Figure S2), were 0.9849, 0.6155, and
0.7433, respectively, while the corresponding values of the anti-
Xoc CoMFA model were 0.9724, 0.6133, and 0.6645,
control efficiencies E (%) = (L − T)/L × 100
respectively. The high q2, r2, and r2
values (>0.5) implied
Defensive Enzyme Activities Determination. Compound 33
(200 mg/L) was sprayed on rice plants that grew under greenhouse
conditions for 30 days until droplets fell, and the rice plants were
inoculated with Xoc. The plants were treated with BT and water in the
same way as the positive and negative controls, respectively. Rice
samples were collected 1, 3, 5, and 7 days after the bacterial infections.
The activity of superoxide dismutase (SOD), peroxidase (POD),
phenylalanine ammonia-lyase (PAL), and catalase (CAT) was tested
using enzyme assay kits (Suzhou Comin Biotechnology Co., Ltd.,
China).
pred
that the CoMFA models have good predictive abilities. The
residues of the training and test sets in the CoMFA models are
presented in Table S1, while the experimental and predicted
value distribution maps are shown in Figure 2a and b. All of
the residues were close to zero and concentrated near the
straight line, illustrating that the CoMFA models might be
reliable. As shown in Figure 2c, the 3- and 6-positions of the
triazole-thiazole were filled with yellow contours in the steric
4647
J. Agric. Food Chem. 2021, 69, 4645−4654