Expression profiling of transgenes (Cry1Ac and Cry2A) in cotton genotypes under different genetic backgrounds

Transgenic cotton carrying the Cry1Ac gene has revolutionized insect pest control since its adoption, although the development of resistance in insect pests has reduced its efficacy.  After 10 years of cultivating Bacillus thuringiensis (Bt) cotton with a single Cry1Ac gene, growers are on the verge of adopting Bt cotton that carries the double gene (Cry1Ac+Cry2A) due to its better effectiveness against insect pests.  Thus, the current study was designed to evaluate the role of each gene in the effectiveness of Bt cotton carrying the double gene.  The expression levels of the Cry1Ac and Cry2A genes were evaluated in the leaves of 10 genotypes (2 parents and 8 F₁ hybrids) at 30 days after sowing (DAS), while samples of leaves, bolls and flowers were taken from the upper and lower canopies at 70 and 110 DAS.  The F₁ hybrids were developed through reciprocal crosses between two Bt (CKC-1, CKC-2) and two non-Bt (MNH-786, FH-942) parents.  The differential expression of transgenes was evaluated through Enzyme Linked Immuno-Sorbent Assay (ELISA).  The results showed that the MNH786×CKC-1 hybrid had the highest concentrations of Cry1Ac gene at 30 DAS (3.08 µg g^–1) and 110 DAS (1.01 µg g^–1) in leaves.  In contrast, the CKC-2×MNH-786 hybrid showed the lowest concentrations of Cry1Ac gene at 30 DAS (2.30 µg g^–1) and 110 DAS (0.86 µg g^–1).  The F₁ hybrid FH-942×CKC-2 showed the highest concentrations of Cry2A gene at 30 DAS (8.39 µg g^–1) and 110 DAS (7.74 µg g^–1) in leaves, while the CKC-1×MNH-786 hybrid expressed the lowest concentrations of Cry2A gene at 30 DAS (7.10 µg g^–1) and 110 DAS (8.31 µg g^–1).  A comparison between the two stages of plant growth showed that leaves had the highest concentrations at 30 DAS, whereas the lowest concentrations were observed at 110 DAS for both genes in leaves.  When the expression pattern was compared between various plant parts in genotype CKC-2, it was found that leaves had higher concentrations of Cry1Ac (3.12 µg g^–1) and Cry2A (8.31 µg g^–1) at 70 DAS, followed by bolls (Cry1Ac (1.66 µg g^–1) and Cry2A (8.15 µg g^–1)) and flowers (Cry1Ac (1.07 µg g^–1) and Cry2A (7.99 µg g^–1)).  The genotype CKC-2 had higher concentrations of Cry1Ac (3.12 µg g^–1) and Cry2A (8.31 µg g^–1) in the upper canopy but less accumulation (2.66 µg g^–1 of Cry1Ac, 8.09 µg g^–1 of Cry2A) in the lower canopy at 70 DAS.  Similarly, at 110 DAS, the expression levels of Cry1Ac and Cry2A in upper and lower canopy leaves were 1.52 and 7.92 µg g^–1, and 0.99 and 7.54 µg g^–1, respectively.  Hence, the current study demonstrates that different genotypes showed variable expression for both of the Cry1Ac and Cry2A genes during plant growth due to different genetic backgrounds.  The Cry2A gene had three-fold higher expression than Cry1Ac with significant differences in expression in different plant parts.  The findings of this study will be helpful for breeding insect-resistant double-gene genotypes with better gene expression levels of Cry1Ac and Cry2A for sustainable cotton production worldwide.     

Chromosome-level genome assembly of Cylas formicarius provides insights into its adaptation and invasion mechanisms

Cylas formicarius is one of the most important pests of sweet potato worldwide, causing considerable ecological and economic damage.  This study improved the effect of comprehensive management and understanding of genetic mechanisms by examining the functional genomics of C. formicarius.  Using Illumina and PacBio sequencing, this study obtained a chromosome-level genome assembly of adult weevils from lines inbred for 15 generations.  The high-quality assembly obtained was 338.84 Mb, with contig and scaffold N50 values of 14.97 and 34.23 Mb, respectively.  In total, 157.51 Mb of repeat sequences and 11 907 protein-coding genes were predicted.  A total of 337.06 Mb of genomic sequences was located on the 11 chromosomes, accounting for 99.03% of the total length of the associated chromosome.  Comparative genomic analysis showed that C. formicarius was sister to Dendroctonus ponderosae, and C. formicarius diverged from D. ponderosae approximately 138.89 million years ago (Mya).  Many important gene families expanded in the C. formicarius genome were involved in the detoxification of pesticides, tolerance to cold stress and chemosensory system.  To further study the role of odorant-binding proteins (OBPs) in olfactory recognition of C. formicarius, the binding assay results indicated that CforOBP4–6 had strong binding affinities for sex pheromones and other ligands.  The high-quality C. formicarius genome provides a valuable resource to reveal the molecular ecological basis, genetic mechanism, and evolutionary process of major agricultural pests; it also offers new ideas and new technologies for ecologically sustainable pest control.     

MicroRNA-370-5p inhibits pigmentation and cell proliferation by downregulating mitogen-activated protein kinase kinase kinase 8 expression in sheep melanocytes

In mammals, microRNAs (miRNAs) play key roles in multiple biological processes by regulating the expression of target genes.  Studies have found that the levels of miR-370-5p expression differ significantly in the skins of sheep with different hair colors; however, its function remains unclear.  In this study, we investigated the roles of miR-370-5p in sheep melanocytes and found that the overexpression of miR-370-5p significantly inhibited cell proliferation (P<0.01), tyrosinase activity (P=0.001) and significantly reduced (P<0.001) melanin production.  Functional prediction revealed that the 3´-untranslated region (UTR) of MAP3K8 has a putative miR-370-5p binding site, and the interaction between these two molecules was confirmed using luciferase reporter assays.  In situ hybridization assays revealed that MAP3K8 is expressed in the cytoplasm of melanocytes.  The results of quantitative RT-PCR and Western blotting analyses revealed that overexpression of miR-370-5p in melanocytes significantly inhibits (P<0.01) MAP3K8 expression via direct targeting of its 3´ UTR.  Inhibition of MAP3K8 expression by siRNA-MAP3K8 transfection induced a significant inhibition (P<0.01) of melanocyte proliferation and significant reduction (P<0.001) in melanin production, which is consistent with our observations for miR-370-5p.  Target gene rescue experiments indicated that the expression of MAP3K8 in melanocytes co-transfected with miR-370-5p and MAP3K8-cDNA (containing sites for the targeted binding to miR-370-5p) was significantly rescued (P≤0.001), which subsequently promoted significant increases in cell proliferation (P<0.001) and melanin production (P<0.01).  Collectively, these findings indicate that miR-370-5p plays a functional role in inhibiting sheep melanocyte proliferation and melanogenesis by downregulating the expression of MAP3K8.       

Investigation of Aegilopsumbellulatafor stripe rust resistance,heading date,and the contents of iron,zinc, and gluten protein

Aegilops umbellulata (UU) is a wheat wild relative that has potential use in the genetic improvement of wheat.  In this study, 46 Ae. umbellulata accessions were investigated for stripe rust resistance, heading date (HD), and the contents of iron (Fe), zinc (Zn), and seed gluten proteins.  Forty-two of the accessions were classified as resistant to stripe rust, while the other four accessions were classified as susceptible to stripe rust in four environments.  The average HD of Ae. umbellulata was significantly longer than that of three common wheat cultivars (180.9 d vs. 137.0 d), with the exception of PI226500 (138.9 d).  The Ae. umbellulata accessions also showed high variability in Fe (69.74–348.09 mg kg^–1) and Zn (49.83–101.65 mg kg^–1) contents. Three accessions (viz., PI542362, PI542363, and PI554399) showed relatively higher Fe (230.96–348.09 mg kg^–1) and Zn (92.46–101.65 mg kg^–1) contents than the others.  The Fe content of Ae. umbellulata was similar to those of Ae. comosa and Ae. markgrafii but higher than those of Ae. tauschii and common wheat.  Aegilops umbellulata showed a higher Zn content than Ae. tauschii, Ae. comosa, and common wheat, but a lower content than Ae. markgrafii.  Furthermore, Ae. umbellulata had the highest proportion of γ-gliadin among all the species investigated (Ae. umbellulata vs. other species=mean 72.11% vs. 49.37%; range: 55.33–86.99% vs. 29.60–67.91%).  These results demonstrated that Ae. umbellulata exhibits great diversity in the investigated traits, so it can provide a potential gene pool for the genetic improvement of these traits in wheat.     

dep1 improves rice grain yield and nitrogen use efficiency simultaneously by enhancing nitrogen and dry matter translocation

The rice cultivars carrying dep1 (dense and erect panicle 1) have the potential to achieve both high grain yield and high nitrogen use efficiency (NUE).  However, few studies have focused on the agronomic and physiological performance of those cultivars associated with high yield and high NUE under field conditions.  Therefore, we evaluated the yield performance and NUE of two near-isogenic lines (NILs) carrying DEP1 (NIL-DEP1) and dep1-1 (NIL-dep1) genes under the Nanjing 6 background at 0 and 120 kg N ha^–1.  Grain yield and NUE for grain production (NUEg) were 25.5 and 21.9% higher in NIL-dep1 compared to NIL-DEP1 averaged across N treatments and planting years, respectively.  The yield advantage of NIL-dep1 over NIL-DEP1 was mainly due to larger sink size (i.e., higher total spikelet number), grain-filling percentage, total dry matter production, and harvest index.  N utilization rather than N uptake contributed to the high yield of NIL-dep1.  Significantly higher NUEg in NIL-dep1 was associated with higher N and dry matter translocation efficiency, lower leaf and stem N concentration at maturity, and higher glutamine synthetase (GS) activity in leaves.  In conclusion, dep1 improved grain yield and NUE by increasing N and dry matter transport due to higher leaf GS activity under field conditions during the grain-filling period.     

Effects of LPA on the development of sheep in vitro fertilized embryos and attempt to establish sheep embryonic stem cells

Lysophosphatidic acid (LPA) is a small molecule glycerophospholipid, which regulates multiple downstream signalling pathways through G-protein-coupled receptors to achieve numerous functions on oocyte maturation and embryo development.  In this study, sheep in vitro fertilized embryos were applied to investigate the effects of LPA on early embryos development and embryonic stem cell establishment.  At first, the maturation medium containing estrus female sheep serum and synthetic oviduct fluid (SOF) were optimized for sheep IVF, and then the effects of LPA were investigated.  From 0.1 to 10 μmol L^–1, LPA had no significant effect on the cleavage rate (P>0.05), but the maturation rate and blastocyst rate increased dependently with LPA concentration (P<0.05), and the blastocyst morphology was normal.  When the LPA concentration was 15 μmol L^–1, the maturation rate, cleavage rate and blastocyst rate decreased significantly (P<0.05), and the blastocyst exhibited abnormal morphology and could not develop into high-quality blastocyst.  Besides, the exogenous LPA increases the expression of LPAR2, LPAR4, TE-related gene CDX-2 and pluripotency-related gene OCT-4 in sheep early IVF embryos with the raise of LPA concentration from 0.1 to 10 μmol L^–1.  The expression of LPAR2, LPAR4, CDX-2 and OCT-4 from the LPA-0.1 μmol L^–1 to LPA-10 μmol L^–1 groups in early embryos were extremely significant (P<0.05), while the expression of these genes significantly decreased in 15 μmol L^–1 LPA-treated embryos compared with LPA-10 μmol L^–1 group (P<0.05).  The inner cell mass in 15 μmol L^–1 LPA-treated embryos was also disturbed, and the blastocysts formation was abnormal.  Secondly, the sheep IVF blastocysts were applied to establish embryonic stem cells.  The results showed that LPA made the blastocyst inoculated cells grow towards TSC-like cells.  They enhanced the fluorescence intensity and mRNA abundance of OCT-4 and CDX-2 as the concentration increased from 0 to 10 μmol L^–1, while 15 μmol L^–1 LPA decreased OCT-4 and CDX-2 expression in the derived cells.  The expression of CDX-2 and OCT-4 in the blastocyst inoculated cells of LPA-1 μmol L^–1 group and LPA-10 μmol L^–1 group extremely significantly increased (P<0.05), but there was significant decrease in LPA-15 μmol L^–1 group compared with LPA-10 μmol L^–1 group (P<0.05).  Meanwhile, the protein expression of LPAR2 and LPAR4 remarkably increased after treatment of LPA at 10 μmol L^–1 concentration.  This study references the IVF embryo production and embryonic stem cell research of domestic animals.      

Screening and evaluation for antibiosis resistance of the spring wheat accessions to the grain aphid,Sitobion miscanthi (Takahashi) (Hemiptera: Aphididae)

Resistant cultivar deployment is an effective method for cereal aphid management.  Under greenhouse conditions, preliminary antibiosis resistance screening was conducted on 114 Ethiopian and 22 Chinese spring wheat accessions.  After performing a bioassay to determine antibiosis resistance, aphid feeding behaviour and phenolic acid content analyses were performed on the aphid resistant wheat accessions by electrical penetration graph (EPG) and high performance liquid chromatography (HPLC), respectively.  Among the wheat accessions, two high resistances, 27 moderate-resistances, and 35 low-resistances to Sitobion miscanthi were identified.  The antibiosis resistance test showed prolonged pre-adult and pre-reproductive periods, shorter reproductive periods, lower fecundity, an intrinsic rate (r_m) of increase, and a finite rate (λ) of increase of S. miscanthi on Lunxuan 145, Wane, Lunxuan 6, 204511, Lunxuan 103 and 5215 than those on the aphid-susceptible accession Beijing 837.  The changes for the parameters of aphid feeding behaviour, including spending a longer time in the penetration and phloem salivation phases and less time in the phloem sap-feeding phase on the resistant wheat accessions, the aphid resistance may occur during the phloem phase and may be due to mechanicalAdditionally, the HPLC analysis showed higher contents of: 1) ferulic acid in Lunxuan 145, Lunxuan 103 and Lunxuan 6; 2) p-coumaric acid in Lunxuan145; 3) vanillic acid in Lunxuan 145, Wane and Lunxuan 6; 4) syringic acid in Lunxuan 103; and 5) caffeic acid in 5215.  The contents of some phenolic acids within wheat leaves, such as p-courmaric acid and vanillic acid showed significant positive correlation with the duration of aphid development, but negative correlation with the aphid fecundity.  The concentrations of these acids may be the causes of antibiosis resistance to S. miscanthi.  The identification of grain aphid-resistant wheat accessions in our study will be helpful in future breeding program for pest control.     

Biosynthesis of artemisinic acid in engineered Saccharomyces cerevisiae and its attractiveness to the mirid bug Apolygus lucorum

Artemisia annua is an important preferred host of the mirid bug Apolygus lucorum in autumn.  Volatiles emitted from A. annua attract A. lucorum.  Volatile artemisinic acid of A. annua is a precursor of artemisinin that has been widely investigated in the Chinese herbal medicine field.  However, little is known at this point about the biological roles of artemisinic acid in regulating the behavioral trends of A. lucorum.  In this study, we collected volatiles from A. annua at the seedling stage by using headspace solid phase microextraction (HS-SPME).  Gas chromatography-mass spectrometry (GC-MS) analysis showed that approximately 11.03±6.00 and 238.25±121.67 ng h^–1 artemisinic acid were detected in volatile samples and milled samples, respectively.  Subsequently, a key gene for artemisinic acid synthesis, the cytochrome P450 gene cyp71av1, was expressed in engineered Saccharomyces cerevisiae to catalyze the production of artemisinic acid.  After the addition of exogenous artemisinic alcohol or artemisinic aldehyde, artemisinic acid was identified as the product of the expressed gene.  In electroantennogram (EAG) recordings, 3-day-old adult A. lucorum showed significant electrophysiological responses to artemisinic alcohol, artemisinic aldehyde and artemisinic acid.  Furthermore, 3-day-old female bugs were significantly attracted by artemisinic acid and artemisinic alcohol at a concentration of 10 mmol L^–1, whereas 3-day-old male bugs were attracted significantly by 10 mmol L^–1 artemisinic acid and artemisinic aldehyde.  We propose that artemisinic acid and its precursors could be used as potential attractant components for the design of novel integrated pest management strategies to control A. lucorum.     

Azole selenourea disrupted the midgut and caused malformed development of Plutellaxylostella

Chemical insecticides targeting the digestive system of diamondback moth (DBM), Plutella xylostella, have not been developed.  The discovery of an insecticide with novel mode of action is a challenge for the control of DBM.  In this study, a class of selenium- and difluoromethyl-modified azoles (fluoroazole selenoureas, FASU) were designed and synthesized for the control of DBM.  Of these azoles, compound B4 showed the highest insecticidal activity against DBM.  The LC₅₀ of third- and second-instar larvae reached 32.3 and 4.6 μg mL^–1, respectively.  The midgut tissue of larvae was severely disrupted, and the larval intestinal tissue was dotted with unique red spots after treatment with compound B4.  Compound B4 led to disintegration of the peritrophic matrix, swelling of the midgut epithelium, fracture of the microvilli, and extensive leakage of cellular debris in the midgut lumen.  Surviving larvae grew very slowly, and the larval duration was significantly prolonged after exposure to compound B4 at sublethal doses (LC₁₀, LC₂₅ and LC₅₀).  Furthermore, the pupation rate, emergence rate and pupae weight were significantly decreased.  Compound B4 also induced abnormal pupae, causing adults to be trapped in the cocoon or failure to fly due to twisted wings.  These results demonstrated that FASU could reduce the population of DBM in sublethal doses.  FASU is the first synthetic insecticidal lead compound that has been shown to disrupt the midgut tissue of the larvae of DBM, and its mode of action totally differs from that of commercial chemical insecticides.     

Diamide derivatives containing a trifluoromethylpyridine skeleton: Design,synthesis, and insecticidal activity

Diamide derivatives are biologically active molecules that have been widely applied in recent years in research on pesticides, especially insecticides.  Using a simple and environmentally friendly scheme, a series of new diamide derivatives containing a trifluoromethylpyridine skeleton was designed, synthesized, and confirmed by ¹H, ¹⁹F and ¹³C NMR, and HR-MS.  Their insecticidal activities against Plutella xylostella and Helicoverpa armigera were measured and the relationship between structure and activity was investigated.  Eight of the title compounds (D2, D5, D10, D21, D28, D29, D30 and D33) showed 100% activity against P. xylostella at 500 mg L^–1.  One compound, D33, still showed 100% activity against P. xylostella at 100 mg L^–1 and had the lowest LC₅₀ (lethal concentration 50%, 3.7 mg L^–1) among the synthesized compounds.  Molecular docking analysis revealed that D33 could be thoroughly embedded in the active pocket of the ryanodine receptor via hydrogen bonding in a manner similar to the commercial insecticide chlorantraniliprole.     

Transcriptional profiling between yellow- and black-seeded Brassica napus reveals molecular modulations on flavonoid and fatty acid content

Brassica napus is an important cash crop broadly grown for the vegetable and oil values.  Yellow-seeded B. napus is preferred by breeders due to its improved oil and protein quality, less pigments and lignin compared with the black-seeded counterpart.  This study compared the differences in flavonoid and fatty acid contents between yellow rapeseed from the progenies of B. napus–Sinapis alba somatic hybrids and the black-seeded counterpart using RNA-seq analysis.  Through HPLC-PDA-ESI(−)/MS² analysis, it was found that phenylpropanoids and flavonoids (i.e., isorhamnetin, epicatechin, kaempferol, and other derivatives) in yellow seed were significantly lower than those in black seed.  The fatty acid (FA) content in yellow rapeseed was higher than that in black rapeseed due to the variation of C16:0, C18:0, C18:1, C18:2, and C18:3 contents.  RNA-seq analysis of seeds at four and five weeks after flowering (WAF) indicated that differentially expressed genes (DEGs) between black and yellow rapeseeds were enriched in flavonoid and FA biosynthesis, including BnTT3, BnTT4, BnTT18, and BnFAD2.  Also, genes related to FA biosynthesis, desaturation and elongation (FAD3, LEC1, FUS3, and LPAT2) in yellow seed were up-regulated compared to those in black seed, while genes involved in beta-oxidation cycle (AIM1 and KAT2) of yellow seed were down-regulated compared to those in black seed.  The DEGs related to the variation of flavonoids, phenylpropanoids, and FAs would help improve the knowledge of yellow seed character in B. napus and promote rapeseed improvement.     

Functional analysis of MdSUT2.1, a plasma membrane sucrose transporter from apple

Sugar content is a determinant of apple (Malus×domestica Borkh.) sweetness.  However, the molecular mechanism underlying sucrose accumulation in apple fruit remains elusive.  Herein, this study reported the role of the sucrose transporter MdSUT2.1 in the regulation of sucrose accumulation in apples.  The MdSUT2.1 gene encoded a protein with 612 amino acid residues that could be localized at the plasma membrane when expressed in tobacco leaf protoplasts.  MdSUT2.1 was highly expressed in fruit and was positively correlated with sucrose accumulation during apple fruit development.  Moreover, complementary growth assays in a yeast mutant validated the sucrose transport activity of MdSUT2.1.  MdSUT2.1 overexpression in apples and tomatoes resulted in significant increases in sucrose, fructose, and glucose contents compared to the wild type (WT).  Further analysis revealed that the expression levels of sugar metabolism- and transport-related genes SUSYs, NINVs, FRKs, HXKs, and TSTs increased in apples and tomatoes with MdSUT2.1 overexpression compared to WT.  Finally, unlike the tonoplast sugar transporters MdTST1 and MdTST2, the promoter of MdSUT2.1 was not induced by exogenous sugars.  These findings provide valuable insights into the molecular mechanism underlying sugar accumulation in apples.     

OsMas1, a novel maspardin protein gene,confers tolerance to salt and drought stresses by regulating ABA signaling in rice

Drought and salt stresses, the major environmental abiotic stresses in agriculture worldwide, affect plant growth, crop productivity, and quality.  Therefore, developing crops with higher drought and salt tolerance is highly desirable.  This study reported the isolation, biological function, and molecular characterization of a novel maspardin gene, OsMas1, from rice.  The OsMas1 protein was localized to the cytoplasm.  The expression levels of OsMas1 were up-regulated under mannitol, PEG6000, NaCl, and abscisic acid (ABA) treatments in rice.  The OsMas1 gene was introduced into the rice cultivar Zhonghua 11 (wild type, WT).  OsMas1-overexpression (OsMas1-OE) plants exhibited significantly enhanced salt and drought tolerance; in contrast, OsMas1-interference (OsMas1-RNAi) plants exhibited decreased tolerance to salt and drought stresses, compared with WT.  OsMas1-OE plants exhibited enhanced hypersensitivity, while OsMas1-RNAi plants showed less sensitivity to exogenous ABA treatment at both germination and post-germination stages.  ABA, proline and K⁺ contents and superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and photosynthesis activities were significantly increased.  In contrast, malonaldehyde (MDA), hydrogen peroxide (H₂O₂), superoxide anion radical (O₂^-·), and Na⁺ contents were significantly decreased in OsMas1-OE plants compared with OsMas1-RNAi and WT plants.  Overexpression of OsMas1 up-regulated the genes involved in ABA signaling, proline biosynthesis, reactive oxygen species (ROS)-scavenging system, photosynthesis, and ion transport under salt and drought stresses.  Our results indicate that the OsMas1 gene improves salt and drought tolerance in rice, which may serve as a candidate gene for enhancing crop resistance to abiotic stresses.     

In vitro and in silico studies of salicylic acid on systemic induced resistance against bacterial leaf blight disease and enhancement of crop yield

Salicylic acid (SA) is an effective elicitor to promote plant defenses and growth.  This study aimed to investigate rice (Oryza sativa L.) cv. Khao Dawk Mali 105 treated with salicylic acid (SA)-Ricemate as an enhanced plant protection mechanism against bacterial leaf blight (BLB) disease caused by Xanthomonas oryzae pv. oryzae (Xoo).  Results indicated that the use of SA-Ricemate as a foliar spray at concentrations of more than 100 mg L^–1 can reduce the severity of BLB  by 71%.  SA-Ricemate treatment also increased the hydrogen peroxide (H₂O₂) content of rice leaf tissues over untreated samples by 39–61%.  Malondialdehyde (MDA) in rice leaves treated with SA-Ricemate also showed an increase of 50–65% when comparing to non-treated samples.  The differential development of these defense compounds was faster and distinct when the SA-Ricemate-treated rice was infected with Xoo, indicating plant-induced resistance.  Besides, SA-Ricemate elicitor at a concentration of 50–250 mg L^–1 was correlated with a substantial increase in the accumulation of total chlorophyll content at 2.53–2.73 mg g^–1 of fresh weight which suggests that plant growth is activated by SA-Ricemate.  The catalase- and aldehyde dehydrogenase-binding sites were searched for using the CASTp server, and the findings were compared to the template.  Chemsketch was used to design and optimize SA, which was then docked to the catalase and aldehyde dehydrogenase-binding domains of the enzymes using the GOLD 3.0.1 Software.  SA is shown in several docked conformations with the enzymes catalase and aldehyde dehydrogenase.  All three catalase amino acids (GLN7, VAL27, and GLU38) were discovered to be involved in the creation of a strong hydrogen bond with SA when SA was present.  In this mechanism, the aldehyde dehydrogenase amino acids LYS5, HIS6, and ASP2 were all implicated, and these amino acids created strong hydrogen bonds with SA.  In field conditions, SA-Ricemate significantly reduced disease severity by 78% and the total grain yield was significantly increased which was an increase of plant height, tiller per hill, and panicle in three field trials during Aug–Nov 2017 and 2018.  Therefore, SA-Ricemate can be used as an alternative elicitor on replacing harmful pesticides to control BLB disease with a high potential of increasing rice defenses, growth, and yield components.     

Study on PCR rapid molecular detection technique of Meloidogyne vitis

Meloidogyne vitis is a new root-knot nematode parasitic on grape root in Yunnan Province, China.  In order to establish a rapid, reliable and specific molecular detection method for M. vitis, the species-specific primers were designed with rDNA-ITS (ribosomal DNA internal transcribed spacer) gene fragment as the target.  The reaction system was optimized and the reliability, specificity and sensitivity of primer were testified, therefore, a rapid PCR detection method for M. vitis was established.  The result showed that the optimal annealing temperature of the primers was 53°C, which was suitable for the detection of different life stages of M. vitis.  Specificity test showed that the specific fragment size of 174 bp was obtained from M. vitis, but other five non-target nematodes did not have any amplification bands, thus effectively distinguish M. vitis and the other five species, and could specifically detect the M. vitis from mixed populations.  Sensitivity test showed that this PCR technique could detect the DNA of a single second-stage juvenile (J₂) and 10^–4 female.  Futhermore, this PCR technique could be used to detect directly M. vitis from soil samples.  The rapid, sensitive and specific PCR molecular detection technique could be used for the direct identification of a single J₂ of M. vitis and the detection of M. vitis in mixed nematode populations and the detection of two J₂s or one male in 0.5 g soil samples, which will provide technical support for the investigation of the occurrence and damage of M. vitis and the formulation of efficient green control strategies.     

Nonphytotoxic copper oxide nanoparticles are powerful “nanoweapons” that trigger resistance in tobacco against the soil-borne fungal pathogen Phytophthora nicotianae

Investigations into the potential application of nanoparticles acting as nanofungicides in sustainable agriculture are rapidly expanding due to the high antimicrobial properties of these compounds, which do not risk inducing pathogen resistance to fungicides.  A detailed understanding of the impact of copper oxide nanoparticles (CuO NPs) on soil-borne phytopathogenic fungi is yet to be obtained.  This study aimed to explore the in vitro antifungal activity and control efficacy of CuO NPs applied via irrigation with respect to tobacco black shank (TBS) disease caused by Phytophthora nicotianae.  The results revealed that CuO NPs greatly interfered with the reproductive growth process of this fungus, repressing hyphal growth, spore germination and sporangium production.  Additionally, morphological damage, intracellular ROS accumulation and increased SOD enzyme activity in hyphae were the antifungicidal mechanisms of these NPs.  In pot experiments, treatment with CuO NPs at 100 mg L^–1 significantly suppressed TBS development, compared with the effect on control plants, and the control efficacy reached 33.69% without inducing phytotoxicity.  Exposure to CuO NPs significantly activated a series of defense enzymes, and resistance genes in tobacco can further explain the mechanisms by which CuO NPs suppressed fungal infection.  The Cu content in both the leaves and roots of P. nicotianae-infested plants increased by 50.03 and 27.25%, respectively, after treatment with 100 mg L^–1 CuO NPs, compared with that of healthy plants.  In particular, a higher Cu content was observed in infected roots than in leaves.  Therefore, this study showed the potential of CuO NPs applied as nanofungicides and as nanoinducers of fungus resistance genes for the management of TBS through inhibition of pathogen infection and stimulation of plant defenses.