Metabolic pathways modulated by coumarin to inhibit seed germination and early seedling growth in Eleusine indica.

Coumarin is an allelochemical that is widely present in the plant kingdom and has great potential for weed control. However, its mechanisms of action remain largely unknown. This study employed metabolomic and transcriptomic analyses along with evaluations of amino acid profiles and related physiological indicators to investigate how coumarin inhibits the germination and seedling growth of Eleusine indica by modifying metabolic pathways. At 72 h of germination at 50 and 100 mg L-1 coumarin, E. indica had lower levels of soluble sugar and activities of amylases and higher levels of starch, O2-, H2O2, auxin (IAA) and abscisic acid (ABA) compared to the control. Metabolomic analysis demonstrated that coumarin treatments had a significant impact on the pathways associated with amino acid metabolism and transport and aminoacyl-tRNA biosynthesis. Exposure to coumarin induced significant alterations in the levels of 19 amino acids, with a decrease in 15 of them, including Met, Leu and γ-aminobutyric acid (GABA). Additionally, transcriptomic analysis showed that coumarin significantly disrupted several essential biological processes, including protein translation, secondary metabolite synthesis, and hormone signal transduction. The decrease in TCA cycle metabolite (cis-aconitate, 2-oxoglutarate, and malate) contents was associated with the suppression of transcription for related enzymes. Our findings indicate that the inhibition of germination and growth in E. indica by coumarin involves the suppression of starch conversion to sugars, modification of the amino acid profile, interference of hormone signalling and the induction of oxidative stress. The TCA cycle appears to be one of the most essential pathways affected by coumarin.

Subtelomeric 5-enolpyruvylshikimate-3-phosphate synthase copy number variation confers glyphosate resistance in Eleusine indica.

Genomic structural variation (SV) has profound effects on organismal evolution; often serving as a source of novel genetic variation. Gene copy number variation (CNV), one type of SV, has repeatedly been associated with adaptive evolution in eukaryotes, especially with environmental stress. Resistance to the widely used herbicide, glyphosate, has evolved through target-site CNV in many weedy plant species, including the economically important grass, Eleusine indica (goosegrass); however, the origin and mechanism of these CNVs remain elusive in many weed species due to limited genetic and genomic resources. To study this CNV in goosegrass, we present high-quality reference genomes for glyphosate-susceptible and -resistant goosegrass lines and fine-assembles of the duplication of glyphosate's target site gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). We reveal a unique rearrangement of EPSPS involving chromosome subtelomeres. This discovery adds to the limited knowledge of the importance of subtelomeres as genetic variation generators and provides another unique example for herbicide resistance evolution.

Temperature influences glyphosate efficacy on glyphosate-resistant and -susceptible goosegrass (Eleusine indica).

Glyphosate has been widely used to control Eleusine indica and other weeds in South China for many years. Among the most troublesome weeds in South China, E. indica can remain alive all year round. However, the influence of temperature on glyphosate efficacy on E. indica, especially under days with fluctuating temperature, is unknown. This study evaluated the influence of two temperature regimes on glyphosate efficacy on glyphosate-resistant (R) and -susceptible (S) E. indica biotypes. Plants of the R and S biotypes were cultivated under two temperature regimes (high: 30°C/20°C day/night; low: 20°C/15°C day/night). Dose-response experiments showed improved efficacy of glyphosate at the low temperature compared with that at the high temperature for both biotypes. Based on the LD50 values, the R biotype was 8.9 times more resistant to glyphosate than the S biotype at the high temperature; however, the resistance index (R/S) decreased to 3.1 at the low temperature. At 4 days after glyphosate application, shikimic acid accumulation was greater at the low temperature than at the high temperature in plants of both biotypes, and the increase was higher in plants of the R biotype than in the S biotype. At a sublethal glyphosate dose (R: 400 g ai ha-1; S: 200 g ai ha-1), plants grown at the low temperature showed a strong decrease in leaf chlorophyll content and Fv/Fm value compared with those of plants grown at the high temperature and the untreated control. At 3 days after treatment, glyphosate absorption was similar between biotypes at the high temperature, but absorption decreased to 64.9% and 53.1% at the low temperature for the R and S biotypes, respectively. For both biotypes, glyphosate translocation from the leaf to the remainder of the plant was reduced at the low temperature compared with that at the high temperature. No differences in glyphosate translocation were observed between biotypes within each temperature regime. This is the first report on the effect of temperature on glyphosate efficacy on E. indica, and provides important insights for glyphosate application and resistance management.

Subtelomeric 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) copy number variation confers glyphosate resistance in Eleusine indica.

Genomic structural variation (SV) can have profound effects on an organism’s evolution, often serving as a novel source of genetic variation. Gene copy number variation (CNV), a specific form of SV, has repeatedly been associated with adaptive evolution in eukaryotes, especially to biotic and abiotic stresses. Resistance to the most widely used herbicide, glyphosate, has evolved through target-site CNV in many weedy plant species, including the economically important cosmopolitan grass, Eleusine indica (goosegrass); however, the origin and mechanisms of these resistance CNVs remain elusive in many weed species due to limited genetic and genomics resources. In order to study the target site CNV in goosegrass, we generated high-quality reference genomes for both glyphosate-susceptible and -resistant individuals, fine assembled the duplication of glyphosate's target site gene enolpyruvylshikimate-3-phosphate synthase (EPSPS), and revealed a novel rearrangement of EPSPS into the subtelomeric region of the chromosomes, ultimately leading to herbicide resistance evolution. This discovery adds to the limited knowledge of the importance of subtelomeres as rearrangement hotspots and novel variation generators as well as provides an example of yet another unique pathway for the formation of CNVs in plants.

A naturally evolved mutation (Ser59Gly) in glutamine synthetase confers glufosinate resistance in plants.

Glufosinate is an important and widely used non-selective herbicide active on a wide range of plant species. Evolution of resistance to glufosinate in weedy plant species (including the global weed Eleusine indica) is underway. Here, we established the molecular basis of target site glufosinate resistance in Eleusine indica. Full-length E. indica glutamine synthetase (GS) iso-genes (EiGS1-1, 1-2, 1-3, and EiGS2) were cloned, and expression of EiGS1-1 and EiGS1-2 was higher than that of EiGS2. A novel point mutation resulting in a Ser59Gly substitution in EiGS1-1 was identified in glufosinate-resistant plants. Rice calli and seedlings transformed with the mutant EiGS1-1 gene were resistant to glufosinate. Purified mutant EiGS1-1 expressed in yeast was more tolerant to glufosinate than the wild-type variant. These transgenic results correlate with a more glufosinate-resistant GS in the crude tissue extract of resistant versus susceptible E. indica plants. Structural modelling of the mutant EiGS1-1 revealed that Ser59 is not directly involved in glufosinate binding but is in contact with some important binding residues (e.g. Glu297) and especially with Asp56 that forms an intratoroidal contact interface. Importantly, the same Ser59Gly mutation was also found in geographically isolated glufosinate-resistant populations from Malaysia and China, suggesting parallel evolution of this resistance mutation.

Evolution of multiple target-site resistance mechanisms in individual plants of glyphosate-resistant Eleusine indica from China.

BACKGROUND: Glyphosate has been used for weed control in South China in various situations for four decades, and most Eleusine indica populations are suspected to have evolved resistance to glyphosate. This research investigated underling target-site glyphosate resistance mechanisms in six field-collected, putative glyphosate-resistant (R) E. indica populations. RESULTS: The six R E. indica populations were confirmed to be low (1.8 to 2.6-fold) to moderately (5.6- to 8.4-fold) resistant to glyphosate relative to the susceptible (S) population. Sixty-seven glyphosate-surviving plants from the six R populations were used to examine target-site resistance mechanisms. Target-site 5-enolpyruvylshikimate3-phosphate synthase (EPSPS) overexpression (OE) (plus further induction by glyphosate treatment) and gene copy number variation (CNV) occurred in 94% R plants, and among them, 16% had the P106A mutation and 49% had the heterozygous double TIPS (T102I + P106S) mutation (plus P381L). In addition, a low number of R plants (6%) only had the homologous TIPS (plus P381L) mutation. The (CT)6 insertion mutation in the EPSPS 5†-UTR always associates with EPSPS OE and CNV. Progeny plants possessing EPSPS OE/CNV (and P106A) displayed low level (up to 4.5-fold) glyphosate resistance. In contrast, plants homozygous for the TIPS mutation displayed higher (25-fold) resistance to glyphosate and followed by plants heterozygous for this mutation plus EPSPS OE/CNV (12-fold). CONCLUSIONS: Target-site glyphosate resistance in E. indica populations from South China is common with prevalence of EPSPS OE/induction/CNV conferring low level resistance. Individual plants acquiring both the TIPS mutation and EPSPS OE/CNV are favored due to evolutionary advantages. The role of (CT)6 insertion mutation in EPSPS CNV is worth further investigation. © 2021 Society of Chemical Industry.

[Allelopathic interactions between Borreria latifolia and two common Asteraceae species]. / 阔叶丰花草与2种菊科植物之间的化感作用.

Borrelia latifolia is an annual herb suitable to be used as cover crop for weed control in orchards in southern China. To understand the competition between B. latifolia and common weeds in orchards, we investigated the allelopathic interactions between B. latifolia and two Asteraceae species, Ageratum conyzoides and Bidens alba. The results showed that the aqueous extracts of B. latifolia at 10-50 mg·mL-1 significantly inhibited seed germination and seedling growth of A. conyzoides and B. alba, with the radicle length of A. conyzoides and B. alba being reduced by 57.4%-90.2% and 57.3%-62.3%, respectively. The aqueous extracts of A. conyzoides and B. alba also had strong allelopathic effects on seed germination of B. latifolia. Under the treatment of 50 mg·mL-1 aqueous extracts, the two Asteraceae species almost entirely inhibited the germination of B. latifolia. After treated with 10 mg·mL-1 aqueous extract of B. latifolia for 30 d, net photosynthesis, plant height and biomass of A. conyzoide were reduced by 15.2%, 20.6% and 41.5%, respectively, compared with the control, while the biomass of B. alba also showed a decreasing trend. Instead, the growth of B. latifolia was not affected. Under mixed culture, biomass of B. latifolia was comparable to that under monoculture, whereas those of A. conyzoides and B. alba were reduced by 86.0% and 27.1%, respectively. Compared with A. conyzoides and B. alba, the allelopathic advantage of B. latifolia is that it can inhibit growth of the two Asteraceae species as well as inhibit seed germination.

Seasonal variations in group leaf characteristics in species with red young leaves.

The leaves of many plants are red during particular stages of their lives, but the adaptive significance of leaf colouration is not yet clearly understood. In order to reveal whether anthocyanins play a similar role (i.e. antioxidants) in different seasonal contexts, this study investigated species with red young leaves in the subtropical forest of Dinghushan biosphere reserve (South China) during summer and winter and compared group leaf characteristics between the two seasons. Of 62 total species, 33 exhibited red young leaves in summer only, 6 in winter only, and 23 in both seasons. The anthocyanins extracted from most of these species had an absorption peak at ~530 nm. Frequency distribution analysis showed that the species containing anthocyanins at levels ranging from 0.02 to 0.04 µmol cm-2 occurred most frequently in summer or winter. Based on conditional grouping of the species, no significant variation was observed in the average anthocyanin contents and antioxidant abilities between summer and winter; the flavonoid content in summer was 2-fold that in winter, whereas the anthocyanin:flavonoid ratio in summer was only half that in winter. Moreover, a positive correlation between anthocyanins and flavonoids was found in summer. Therefore, it is less likely for anthocyanins to serve as antioxidants in summer than winter, because such a function in summer leaves is readily replaced by other flavonoids.

Flooding with shallow water promotes the invasiveness of Mikania micrantha.

The invasive ability of alien plants is not only affected by their biological characteristics but also by environmental factors. Therefore, investigating the relationship between plant growth and environmental factors is helpful for predicting the invasive potential of alien species. Mikania micrantha H.B.K. (a vine of Asteraceae) is one of the top 10 most invasive weeds worldwide and causes serious damage to agroforestry ecosystems. Water is an important environmental factor that affects plant growth; however, the relationship between water conditions and the rapid growth of M. micrantha is not clear. In this study, 162 M. micrantha population sizes were investigated in dry, wet and aquatic habitats in the Pearl River Delta region of Guangdong, China. In addition, the seed germination and seedling growth characteristics of M. micrantha were determined by submerging tests. The results showed that the population size of M. micrantha was the largest in aquatic habitats, and the soil moisture content was positively correlated to the population size in dry and wet habitats. Furthermore, M. micrantha seeds could germinate underwater and grow out of the water surface at a depth of 6 cm with a survival rate of 7.4%. Aquatic habitat promoted vine elongation, whereas dry habitats resulted in the reverse pattern. After 8 weeks of water treatments, the vine stem length was 2 and 3 times longer in the aquatic habitat than the wet and dry habitats, respectively. The total root length, root volume, and root tip number increased significantly in the aquatic habitat when compared to those in the wet habitat; however, these parameters exhibited the opposite pattern in the dry habitat. The results showed that flooding with shallow water is conducive to the invasiveness of M. micrantha, suggesting that water is the key determinant during the intrusion process of M. micrantha populations. OPEN RESEARCH BADGES: This article has been awarded Open Data, Open Materials and Preregistered research design Badges. All materials and data are publicly accessible via the Open Science Framework at https://osf.io/ksz2f/?viewonly=30b6fec21f0447edbdfc9cebe2b01065, https://osf.io/a5ymf/ and https://osf.io/ksz2fl?viewonly=cfcbfOfc829c402fb22deb3be801dffc.

Fenoxaprop-P-ethyl and mesosulfuron-methyl resistance status of shortawn foxtail (Alopecurus aequalis Sobol.) in eastern China.

Resistance to the acetyl-coenzyme A carboxylase (ACCase)- and acetolactate synthase (ALS)- inhibiting herbicides in shortawn foxtail (Alopecurus aequalis) has been reported in wheat fields of eastern China. To better understand the distribution of the resistant populations and the occurrence of the target-site mutations, 74 populations collected from Anhui, Jiangsu and Shandong province were surveyed, and the ACCase and ALS gene fragments, encompassing all the documented mutant codon positions, were amplified and sequenced. Plants from 37 and 34 populations survived fenoxaprop-P-ethyl and mesosulfuron-methyl treatment at 62.1 g a.i. ha-1 and 9 g a.i. ha-1 respectively, with different survival rates. Twenty-seven populations exhibited multiple resistance to fenoxaprop-P-ethyl and mesosulfuron-methyl. Whole-plant dose-response experiments showed that the resistance index ranged from 6.2 to 167.8 for fenoxaprop-P-ethyl, and from 7.8 to 139.5 for mesosulfuron-methyl. Four ACCase (I1781L, I2041N, I2041T and D2078G) and four ALS (P197R, P197S, P197T and W574 L) resistance mutations were detected respectively. Individuals containing two amino acid substitutions were also found. D2078G and W574 L were predominant ACCase and ALS gene mutations respectively. This study has shown that fenoxaprop-P-ethyl and mesosulfuron-methyl resistance was prevalent in A. aequalis in eastern China, and target site mutations in the ACCase and ALS gene were one of the most common mechanisms.

Alterations in the 5' untranslated region of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene influence EPSPS overexpression in glyphosate-resistant Eleusine indica.

BACKGROUND: The herbicide glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Overexpression of the EPSPS gene is one of the molecular mechanisms conferring glyphosate resistance in weeds, but the transcriptional regulation of this gene is poorly understood. The EPSPS gene was found to be significantly up-regulated following glyphosate treatment in a glyphosate-resistant Eleusine indica population from southern China. To further investigate the regulation of EPSPS overexpression, the promoter of the EPSPS gene from this E. indica population was cloned and analyzed. RESULTS: Two upstream regulatory sequences, Epro-S (862 bp) and Epro-R (877 bp), of EPSPS were obtained from glyphosate-susceptible (S) and -resistant (R) E. indica plants, respectively, by high-efficiency thermal asymmetric interlaced polymerase chain reaction (HiTAIL-PCR). The Epro-S and Epro-R sequences were 99% homologous, except for two insertions (3 and12 bp) in the R sequence. The 12-base insertion in the Epro-R sequence was located in the 5' untranslated region (UTR) pyrimidine nucleotide-rich (Py-rich) stretch element. Promoter activity tests showed that the 12-base insertion resulted in significant enhancement of Epro-R promoter activity, whereas the 3-base insertion had little effect on Epro-R promoter activity. CONCLUSION: Alterations in the 5' UTR Py-rich stretch element of EPSPS are responsible for glyphosate-induced EPSPS overexpression. Thus, EPSPS transcriptional regulation confers glyphosate resistance in this E. indica population. © 2018 Society of Chemical Industry.

Functional characteristics of phenolic compounds accumulated in young leaves of two subtropical forest tree species of different successional stages.

The abundance of phenolic compounds (including anthocyanins) in leaves is associated with photosynthetic performance, but the regulatory mechanism is unclear. Schima superba Gardn. et Champ. and Cryptocarya concinna Hance., which exhibit distinct anthocyanin accumulation patterns, are dominant tree species in the early- and late-successional stages, respectively, of subtropical forests in China. RNA-seq and analyses of phenolic concentrations, antioxidant capacity and photosynthetic characteristics were performed on young and mature leaves of these two species under contrasting light conditions. The high-light-acclimated young leaves of S. superba and C. concinna and low-light-acclimated young leaves of C. concinna were red. These red leaves had higher ratios of electron transport rate to gross photosynthesis (ETR:Pgross) and total antioxidant capacity to chlorophyll (TAC:Chl) than did the green leaves, regardless of light conditions. In addition, the red leaves had a higher expression level of the UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene than did the green leaves, irrespective of light conditions. Total antioxidant capacity was positively correlated with flavonoid content in C. concinna leaves and with total phenolic content in leaves of both species under both high and low light. Consistent with the measurements of photosynthetic performance and flavonoids:Chl ratio, photosynthesis-related genes were extensively downregulated and flavonoid-pathway-related genes were extensively upregulated in young leaves relative to mature leaves. Under high and low light, both non-photochemical quenching and TAC:Chl, which serve as different types of photoprotective tools, were enhanced in young leaves of S. superba, whereas only TAC:Chl was enhanced in young leaves of C. concinna. Our results indicate that the biosynthesis of phenolic compounds in young leaves is likely enhanced by an imbalance between photosynthetic electron supply and demand and that flavonoids play a larger role in meditating photoprotection in late-successional species than in early-successional ones.

A new strategy for controlling invasive weeds: selecting valuable native plants to defeat them.

To explore replacement control of the invasive weed Ipomoea cairica, we studied the competitive effects of two valuable natives, Pueraria lobata and Paederia scandens, on growth and photosynthetic characteristics of I. cairica, in pot and field experiments. When I. cairica was planted in pots with P. lobata or P. scandens, its total biomass decreased by 68.7% and 45.8%, and its stem length by 33.3% and 34.1%, respectively. The two natives depressed growth of the weed by their strong effects on its photosynthetic characteristics, including suppression of leaf biomass and the abundance of the CO2-fixing enzyme RUBISCO. The field experiment demonstrated that sowing seeds of P. lobata or P. scandens in plots where the weed had been largely cleared produced 11.8-fold or 2.5-fold as much leaf biomass of the two natives, respectively, as the weed. Replacement control by valuable native species is potentially a feasible and sustainable means of suppressing I. cairica.

Enhancement of mucosal immune responses by intranasal co-delivery of Newcastle disease vaccine plus CpG oligonucleotide in SPF chickens in vivo.

Immunostimulatory CpG oligodeoxynucleotides (ODN) have been tested as immunoadjuvants for various vaccines in mice and human. Findings from previous reports suggest that CpG ODN can be used to enhance magnitude and balance of an immune response while reducing undesirable side effects of commercial vaccine, when delivered by parenteral route. Recently, it has been showed that CpG ODN is a promising mucosal adjuvant in mice, but data on mucosal immune responses induced by CpG ODN in other animals, especially in chickens, are scarce. Herein, we evaluated intranasal (IN) delivery of CpG ODN with newcastle disease (ND) vaccine (NDV) to determine its potential as a mucosal adjuvant to a commercial vaccine. CpG ODN augmented systemic (IgG in serum, T cell proliferation) and mucosal (IgA in intestinal washings and feces) immune responses against antigen. CpG ODN stimulated effectively both systemic and mucosal immune responses when delivered intranasally. Results from this study indicate that stimulatory CpG ODN is a potential effective mucosal adjuvant for the NDV in SPF chickens and may be applicable to husbandry animals.

Intranasal administration of CpG oligonucleotides induces mucosal and systemic Type 1 immune responses and adjuvant activity to porcine reproductive and respiratory syndrome killed virus vaccine in piglets in vivo.

Oligonucleotides containing CpG motifs (CpG ODN) are strong adjuvants for immune responses, particularly in mice. Recently, it has been showed that CpG ODN is a promising mucosal adjuvant in mice, but data on mucosal immune responses induced by CpG ODN in piglets are scarce. We have previously demonstrated that CpG ODN is a potent adjuvant to pseudorabies attenuated virus (PRV) vaccine when administered subcutaneously (SC) in newborn piglets. Herein, we evaluated intranasal (IN) delivery of CpG ODN with porcine reproductive and respiratory syndrome (PRRS) killed virus vaccine (PRRSV) to determine its potential as a mucosal adjuvant to a commercial vaccine. CpG ODN augmented systemic (IgG in serum, Peripheral blood mononuclear cells (PBMC) proliferation) and mucosal (IgA in feces, nasal and oral secretions) immune responses against antigen. CpG ODN stimulated both T-helper type1 (Type 1) (IgG2) and Type 2 (IgA) responses when delivered intranasally. Results from this study indicate that stimulatory CpG ODN may be effective as a mucosal adjuvant with commercial vaccine in husbandry animals.

[The function of plant-associated bacterial genome research on bacteria-plant interactions].

Genomic and post-genomic research is responsible for the accumulation of enormous data, which allows the formation of whole views of the organisms under investigation. Genome research on pathogenic and associative bacteria is providing important information on bacteria-plant interactions, especially on type-III secretion systems (TTSS) and their role in the interaction of bacteria with plants. In the present paper, we summarized plant-associated bacterial genome research on bacteria-plant interactions in details. Trends in future research have also been analyzed.

CpG oligodeoxynucleotides augment the immune responses of piglets to swine Pasteurella multocida living vaccine in vivo.

Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) prevent development of T-helper type 2 (Th2) immune response and reverse established allergic responses in mouse models. However, little work on immune responses in piglets has been conducted in vivo. In this report, the ability of a porcine-specific CpG ODN to act as an immunostimulant and enhance immune responses of piglets to swine Pasteurella multocida living vaccine (SPML vaccine) was determined. The titre of IgG and IgG1/IgG2 isotype to SPML vaccine in serum, the proliferation of lymphocytes, SPML-specific interferon-gamma (IFN-gamma) and IL-6, TNF-alpha, IL-4 production of PBMCs in vitro and IFN-gamma, IL-6, TNF-alpha, IL-4, IL-10 in piglets serum were examined to identify the immune responses of the piglets. Immune responses of the piglets vaccinated with SPML and CpG ODN were significantly stronger than responses of piglets vaccinated with SPML alone. All these data summarized that immunostimulatory CpG ODN could modulate the immune response towards a Th1-like response when co-administered to piglets during SPML vaccination, which suggested that the therapeutic uses envisioned for these ODNs (as vaccine adjuvants and immunoprotective agents) may be applicable to husbandry animals.

In vivo effects of oligodeoxynucleotides containing synthetic immunostimulatory motifs in weaned piglets.

The innate immunostimulatory effects of CpG ODN have been demonstrated in mouse, monkey, sheep and chicken in some reports. Unfortunately, little work has been carried out with regard to their effects on the innate immune system of weaned piglets. In this study, the proportion of CD4(+), CD8(+) T lymphocytes subpopulations and interferon-gamma (IFN-gamma) and IL-4 in serum, proliferation of peripheral blood mononuclear cells (PBMCs) were tested at different time-points. The results suggested that, the CD4(+)/CD8(+) ratio decreased significantly in weaned piglets inoculated with phosphate buffer saline (PBS) alone, however, it was stable in CpG ODN-inoculated piglets. Proliferation of PBMCs and IFN-gamma levels of CpG ODN-injected piglets were significantly higher than those of PBS-injected piglets. The ODN-induced responses were stronger in animals injected with CpG ODN formulated in 30% emulsigen than in PBS and alum. The innate immunostimulatory activity of CpG ODN appeared to be in dose-dependent manner. These in vivo data demonstrate for the first time that CpG ODN can stimulate innate immune system in weaned piglets.