Tannic Acid-Steeped Corn Grain Modulates in vitro Ruminal Fermentation Pattern and Microbial Metabolic Pathways.

This study investigated the effects of tannic acid (TA)-treated corn on changes in ruminal fermentation characteristics and the composition of the ruminal bacterial community in vitro. Ruminal fluid was obtained from three rumen-fistulated goats fed a 60:40 (forage/concentrate) diet. The batch cultures consisted of 25 ml of strained rumen fluid in 25 ml of an anaerobic buffer containing 0.56 g of ground corn, 0.24 g of soybean meal, 0.10 g of alfalfa, and 0.10 g of oat grass. Ground corn (2 mm) was steeped in an equal quantity (i.e., in a ratio of 1:1, w/v) of water alone (Con), 15 (TA15), 25 (TA25), and 35 g/l (TA35) TA solution for 12 h. After incubation for 24 h, TA-treated corn linearly increased (P <0.05) ruminal pH and the molar proportion of acetate, but linearly reduced (P <0.05) total volatile fatty acids and the molar proportion of butyrate compared with the Con treatment. Illumina MiSeq sequencing was used to investigate the profile changes of the ruminal microbes. A principal coordinates analysis plot based on weighted UniFrac values revealed that the structure of the ruminal bacterial communities in the control group was different from that of the TA-treated corn groups. The results of changes in the rumen bacterial communities showed that TA-treated corn linearly enriched (P <0.05) Rikenellaceae_RC9_gut_group, but linearly reduced (P <0.05) Ruminococcaceae_NK4A214_group, Ruminococcus_2, and unclassified_o__Clostridiales. Functional prediction of ruminal microbiota revealed that the TA-treated corn linearly decreased ruminal microbiota function of utilizing starch through pyruvate metabolism. In conclusion, TA-treated corn can modulate the rumen fermentation characteristics, microbial composition, and metabolic pathways, which may be potentially useful for preventing the occurrence of ruminal acidosis.

[Sex difference in height growth and blood pressure change among Beijing school-age children and adolescents: a ten-year longitudinal study].

Objective: To explore sex difference in height growth and blood pressure (BP) change among Beijing school-age children and adolescents. Methods: Using physical examination data of 70 769 school-age children and adolescents from primary to high school during 2009-2018 in Shunyi District, a longitudinal dataset was formed with completed anthropometrical measurements of height and blood pressure (BP) after individual information linkage. Age-specific height, BP, growth rate of height and BP as well BP growth rate based on age at peak height velocity (PHA) were calculated. Linear mixed-effects model was used to identify sex disparity in the growth rates of height and BP. Results: Height and BP increased with age in both boys and girls, and the mean height and BP of boys were always higher than those of girls, except age group from 10 to 11 years. Sex disparity existed in growth rates of height and BP (P<0.001), which was demonstrated by the interaction item of"sex x age"in linear mixed-effects model. The PHA of boys was 12 years old, which was 2 years later than that of girls, about 10 years old. The curves of BP growth rate with age showed double peaks in both boys and girls. Boys reached the peak BP velocity at 13 years old, 3 years lagging behind that of girls who reached the peak at 10 years old. However, the peak of height and BP velocity of boys were higher than that of girls. The change of BP was highly synchronized in time with the increase of height, after adjusting for the growth rate of height by PHA. BP velocity increased with age before onset of puberty till PHA and then declined. Conclusion: Sex disparity in height growth and BP change among school-age children and adolescents is persistent and significant and the change of BP is highly synchronized in time with the increase of height.

[Association of blood pressure and left ventricular mass in children from the PROC cohort study].

Objective: To explore the association between blood pressure (BP) and the left ventricular mass (LVM) in children aged 6-8 years. Methods: The participants were from the community-based census-like design child cohort on sensitization, puberty, obesity and cardiovascular risk (PROC) conducted in six public non-boarding primary schools in Shunyi District, Beijing. Repeated three measurements on anthropometrical, M-mode and 2-dimensional (2D) echocardiographic imaging (2D/M ECHO) and blood biochemical assay, and BP measurements were carried out at baseline and follow-up from October 2018 to June 2019. A total of 1 659 children who had repeated BP measurements and cardiac structure information were included in this study. The average value of last two measurements of BP was determined as BP value for analysis. Formula recommended by Devereux was used to calculate the mass of left ventricle. Robust linear regression models were used to explore the association between BP and LVM in different groups. Results: The average age of all patients was (7.10±0.29) years old, including 832 boys (50.15%). Of all, 83.54% (1 386/1 659) were grouped as normal BP with average LVM (58.54±13.33) g, and 16.46% (273/1 659) as elevated BP group with LVM (63.84±15.78) g (P<0.001). The LVM of the normal BP group was lower than elevated BP group in overall participants, boys and girls (P<0.005). Univariate analysis showed that systolic BP was associated with LVM in overall, boys and girls (P<0.001) respectively. While diastolic BP was associated with LVM in overall and girls (P<0.03). Multivariable analysis indicated that the associations between systolic BP and LVM were observed in overall, boys and girls (P<0.05) with the ß (95%CI)=0.14 (0.08, 0.21), 0.18 (0.08, 0.27) and 0.12 (0.03, 0.22), respectively. However, the associations of diastolic BP and LVM were not significant. Conclusion: Systolic BP is highly associated with LVM and elevated BP could increase the LVM in children. Boys with elevated BP present a larger LVM and might indicate higher risk of left ventricular hypertrophy in adulthood.

[Association between trunk fat index and carotid intima-media thickness among children aged 6-8 years old in Beijing].

Objective: The study is to explore the association between trunk fat index (TFI) and carotid intima-media thickness (cIMT) among children aged 6-8 years old in Shunyi District, Beijing. Methods: The participants were enrolled from the child cohort on sensitization, puberty, obesity and cardiovascular risk (PROC) conducted in Shunyi District, Beijing from October 2018 to June 2019. The PROC used a community-based census-like design, and all eligible first-grade children from six public non-boarding primary schools in urban area were approached. Finally, a total of 1 503 children with written informed consent from parents and had complete data of TFI and cIMT were included for the present study. Sequential baseline surveys including anthropometric measurements, laboratory testing and ultrasonography measurement were conducted to collect the data on height, weight, body composition, blood pressure, serum lipids and cIMT. Linear regression was used to determine the predictors of cIMT, receiver operating characteristic (ROC) curve analysis was used to determine the cut-off value of TFI to identify children with high cIMT, and analysis of covariance was used to evaluate the post-consistency classification of cIMT by TFI. Results: The age of 1 503 participants was (6.7±0.3) years, and 752 boys accounted for 50.0%. The average cIMT was (0.358±0.024) and (0.355±0.023) mm, and the M (P25, P75) of TFI was 0.70 (0.22, 1.78) and 0.74 (0.23, 1.52) kg/m2 for boys and girls, respectively. The detection rates for boys and girls of high cIMT were 2.1% and 3.3%, respectively. Linear regression analysis showed that height, systolic blood pressure (SBP), diastolic blood pressure (DBP), TFI were positively correlated with cIMT in boys (P values<0.05). And height, SBP, triglyceride (TG), TFI were positively correlated with cIMT, and high-density lipoprotein cholesterol (HDL-C) was negatively correlated with cIMT in girls (P values<0.05). ROC curve analysis indicated that the best cut-off values for TFI to identify children with high cIMT were 1.78 and 1.14 kg/m2, at P75 and P66 for boys and girls, respectively. After grouped with the cut-off value of TFI and adjusted for age, height, SBP, DBP, TG, HDL-C, multivariable covariance analysis showed a consistent cut-off of inter-group cIMT mean by TFI groups (P values<0.005). Conclusion: TFI is associated with cIMT, which underscore its application potential in identifying early vascular structural damage.

CREG improves cardiac function by regulating cardiomyocytes' autophagy in diabetic myocardial infarction rats.

OBJECTIVE: We aimed to observe the changes of cardiac function, cell morphology, cellular repressor of E1A-stimulated genes (CREG) and LC3-II after myocardial infarction (MI) in non-diabetic and diabetic rats, and to explore the relationship between myocardial damage and CREG and autophagy in diabetic rats. MATERIALS AND METHODS: Diabetic rat models were prepared by intraperitoneal injection of low concentration (50 mg/kg) streptozotocin (STZ). MI models were established in normal rats and diabetic rats. The cardiac function of each group was detected by echocardiography. The pathological results of myocardial tissue in the infarcted area were observed under light microscope. The expression of CREG was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Myocardial protein CREG LC3-II expression was measured by Western blot. Autophagy levels were also detected at the cellular level. Construction of CREG overexpressing adenovirus transfected H9c2 cells, and injection of rats with AAV-CREG to achieve the purpose of overexpressing CREG. In vitro and in vivo experiments were conducted to explore the effects of CREG on autophagy and cardiac function in a diabetic MI model. RESULTS: Compared with the non-diabetic sham (NS) group, there were no marked differences in cardiac function and CREG levels in the diabetic sham (DS) group. Compared with the NS group, the cardiac function of the non-diabetic myocardial infarction (NI) group and the diabetic infarction myocardial (DI) group were reduced, and the levels of autophagy were increased. However, the cardiac function of the DI group was worse than that of the NI group, and the autophagy level of the DI group was lower than the NI group. The results at the cellular level were similar to the experiments in vivo. The overexpression of CREG in vivo or in vitro can increase autophagy levels and improve cardiac function. CONCLUSIONS: The exacerbation of myocardial injury after MI in diabetic rats may be related to the inhibition of CREG in myocardial cells of infarcted area by diabetes.

[Biological characteristics and genomic information of a bacteriophage against pan-drug resistant Klebsiella pneumoniae in a burn patient and its effects on bacterial biofilm].

Objective: To isolate a bacteriophage against pan-drug resistant Klebsiella pneumoniae in a burn patient, and to study its biological characteristics, genomic information, and effects on bacterial biofilm. Methods: (1) In 2018, pan-drug resistant Klebsiella pneumoniae UA168 (hereinafter referred to as the host bacteria) solution isolated from the blood of a burn patient in Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (hereinafter referred to as Ruijin Hospital) was used to isolate and purify the bacteriophage against pan-drug resistant Klebsiella pneumoniae from the sewage of Ruijin Hospital with sewage co-culture method, drip plate method, and double-agar plate method. The bacteriophage was named as phage KP168 and the plaque morphology was observed. (2) The phage KP168 solution was taken for cesium chloride density gradient centrifugation and dialysis, and then the morphology of phage KP168 was observed through transmission electron microscope after phosphotungstic acid negative staining. (3) The phage KP168 solution was taken to determine the lytic ability of the phage KP168 against 20 strains of pan-drug resistant Klebsiella pneumoniae isolated from the burned patients' blood in Ruijin Hospital by the drip plate method, and then the lysis rate was calculated. (4) The phage KP168 solution at a initial titer of 9.3×10(11) plaque-forming unit (PFU)/mL (400 µL per tube) and the host bacteria solution at a concentration of 1×10(9) colony-forming unit (CFU)/mL (4 mL per tube) were conventionally shaking cultured together for 4 hours at multiplicity of infection (MOI) of 10.000, 1.000, 0.100, 0.010, or 0.001, respectively (1 tube per MOI). The titer of phage KP168 was measured by the double-agar plate method (the measurement method was the same below) to select the optimal MOI. The experiment was repeated three times. (5) The host bacteria solution at a concentration of 1×10(9) CFU/mL (4 mL per tube) and the phage KP168 solution at an adjusted titer of 5×10(7) PFU/mL (400 µL per tube) were mixed at the MOI of 0.005. The plaques were counted 0 (immediately), 1, 2, 3, 4, 5, 15, and 30 minutes (1 tube at each time point) after mixing by the double-agar plate method (the counting method was the same below), and the percentage of adsorbed phages was calculated to screen for the optimal adsorption time. The experiment was repeated three times. (6) The host bacteria solution at a concentration of 1×10(9) CFU/mL (300 µL per tube) and the phage KP168 solution at a titer of 5×10(8) PFU/mL (60 µL per tube) were mixed at MOI of 0.005 and conventionally shaking cultured after standing for the optimal adsorption time. The phage KP168 titer was measured 0 (immediately), 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 minutes after culture, and a one-step growth curve was drawn. The experiment was repeated three times. (7) The phage KP168 solution at a titer of 2.5×10(10) PFU/mL was left to stand for 1 hour at 37, 40, 50, 60, or 70 ℃ (3 tubes at each time point, 1 mL per tube) for counting the plaques, and then the thermal stability curve was drawn. SM buffer at a pH values of 5.0, 6.0, 7.0, 7.4, 8.0, 9.0, or 10.0 were added to the phage KP168 solution at a titer of 3.0×10(10) PFU/mL, respectively. The mixed solution was left to stand for 1 hour at 37 ℃ (3 tubes of each pH, each tube containing 100 µL phage KP168 solution and 900 µL SM buffer), and then the plaques were counted, and an acid-base stability curve was drawn. (8) The phage KP168 solution was taken for DNA extraction and sequencing after dialysis as in experiment (2). The whole genome was annotated with Prokka to obtain the coding sequence of phage KP168. Nucleotide's BLAST function was used to proceed nucleic acid sequence alignment for finding a known phage with the highest similarity to the phage KP168 nucleic acid sequence, and Blastx function was used to translate the coding sequence into protein for its function prediction. The comparison with Antibiotic Resistance Genes Database and Virulence Factors Database was proceeded. (9) In a 96-well plate, at a MOI of 1.000, 0.100, 0.010 or 0.001 (3 wells per MOI), 20 µL phage KP168 solution at a initial titer of 5.8×10(10) PFU/mL was added to 200 µL host bacteria solution at a concentration of 1.5×10(8) CFU/mL (the same concentration below) for co-cultivation for 48 hours. After 200 µL host bacteria solution was left to stand for 48 hours, 20 µL phage KP168 solution at a titer of 1×10(6,) 1×10(7,) 1×10(8,) 1×10(9,) or 1×10(10) PFU/mL (3 wells per titer) was added respectively for action for 4 hours. In both experiments, 200 µL host bacteria solution added with 20 µL SM buffer (3 wells) acted as a negative control, and 220 µL LB culture medium (3 wells) acted as a blank control. Absorbance values were measured by a microplate reader, and inhibition/destruction rates of biofilm were calculated. The experiments were both repeated three times. Results: (1) The plaques of phage KP168 successfully isolated and purified were transparent and round, and its diameter was approximately 1.5 mm. (2) The phage KP168 has a regular polyhedron structure with a diameter of about 50 nm and without a tail. (3) The phage KP168 could lyse 13 of 20 strains of Klebsiella pneumoniae from burned patients, with a lysis rate of 65.0%. (4) When MOI was 1.000, the titer was the highest after co-culturing the phage KP168 with the host bacteria for 4 hours, which was the optimal MOI. (5) After the mixing of the phage KP168 with the host bacteria for 4 minutes, the percentage of the adsorbed phage reached the highest, which was the optimal adsorption time. (6) The one-step growth curve showed that during the lysis of the host bacteria by phage KP168, the incubation period was about 10 minutes, and the lysis period was about 40 minutes. (7) With the condition of 40 ℃ or pH 7.4, the number of plaques and the activity of phage KP168 reached the highest. (8) The genome of phage KP168 was a linear double-stranded DNA with a length of 40 114 bp. There were 48 possible coding sequences. It had the highest similarity to Klebsiella phage_vB_Kp1. The most similar known proteins corresponding to the translated proteins of coding sequences contained 23 hypothetical proteins and 25 proteins with known functions. No resistance genes or virulence factor genes were found. The GeneBank accession number was KT367885. (9) After 48 hours of co-cultivation of the phage KP168 and the host bacteria at each MOI, the inhibition rates of biofilm were similar, with an average of about 45%. After the phage KP168 with a titer of 1×10(9) PFU/mL acted on the biofilm formed by the host bacteria for 4 h, the destruction rate of biofilm was the highest, reaching an average of 42%. Conclusions: In this study, a bacteriophage against pan-drug resistant Klebsiella pneumoniae from a burn patient, phage KP168, is isolated from sewage, which belongs to the tailless phage. It has a wide host spectrum, short adsorption time, and short incubation period, with certain thermal and acid-base stability. Its genomic information is clear, and it does not contain resistance genes or virulence factor genes. It also has an inhibitory effect on the formation of bacterial biofilm and a destructive effect on the formed bacterial biofilm.

Thiamine ameliorates inflammation of the ruminal epithelium of Saanen goats suffering from subacute ruminal acidosis.

This study aimed to examine the role of thiamine in the local inflammation of ruminal epithelium caused by high-concentrate diets. Eighteen mid-lactating (148 ± 3 d in milk; milk yield = 0.71 ± 0.0300 kg/d) Saanen goats (body weight = 36.5 ± 1.99 kg; body condition score = 2.73 ± 0.16, where 0 = emaciated and 5 = obese) in parity 1 or 2 were selected. The goats were randomly divided into 3 groups (n = 6/group): (1) control diet (concentrate:forage 30:70), (2) high-concentrate diet (HC; concentrate:forage 70:30), and (3) high-concentrate diet with 200 mg of thiamine/kg of dry matter intake (THC; concentrate:forage 70:30). Goats remained on experimental diets for 8 wk. On the last day of 8 wk, ruminal and blood samples were collected to determine ruminal parameters, endotoxin lipopolysaccharide, and blood inflammatory cytokines. Goats were slaughtered to collect ruminal tissue to determine gene and protein expression of toll-like receptor 4 (TLR4) signaling pathways. Thiamine supplementation increased ruminal pH (6.03 vs. 5.42) compared with the HC group. Propionate (21.08 vs. 31.61 mM), butyrate (12.08 vs. 19.39 mM), lactate (0.52 vs. 0.71 mM), and free lipopolysaccharide (42.16 vs. 55.87 × 103 endotoxin units/mL) concentrations in ruminal fluid were lower in THC goats compared with HC goats. Similar to plasma interleukin 1ß (IL-1ß) concentration (209.31 vs. 257.23 pg/mL), blood CD8+ percentage (27.57 vs. 34.07%) also decreased in response to thiamine. Compared with HC goats, THC goats had lower ruminal epithelium activity of the enzymes myeloperoxidase and matrix metalloproteinase (MMP) 2 and 9. In contrast to HC, THC had downregulated mRNA expression of nuclear factor-κB (NFKB), TLR4, IL1B, MMP2, and MMP9 in ruminal epithelium. Thiamine supplementation led to lower relative protein expression of IL-1ß, NF-κB unit p65, and phosphorylated NF-κB unit p65 in ruminal epithelium. Taken together, these results suggest that thiamine supplementation mitigates HC-induced local inflammation and ruminal epithelial disruption.

Colletotrichum species associated with anthracnose of Pyrus spp. in China.

Colletotrichum species are plant pathogens, saprobes, and endophytes on a range of economically important hosts. However, the species occurring on pear remain largely unresolved. To determine the morphology, phylogeny and biology of Colletotrichum species associated with Pyrus plants, a total of 295 samples were collected from cultivated pear species (including P. pyrifolia, P. bretschneideri, and P. communis) from seven major pear-cultivation provinces in China. The pear leaves and fruits affected by anthracnose were sampled and subjected to fungus isolation, resulting in a total of 488 Colletotrichum isolates. Phylogenetic analyses based on six loci (ACT, TUB2, CAL, CHS-1, GAPDH, and ITS) coupled with morphology of 90 representative isolates revealed that they belong to 10 known Colletotrichum species, including C. aenigma, C. citricola, C. conoides, C. fioriniae, C. fructicola, C. gloeosporioides, C. karstii, C. plurivorum, C. siamense, C. wuxiense, and two novel species, described here as C. jinshuiense and C. pyrifoliae. Of these, C. fructicola was the most dominant, occurring on P. pyrifolia and P. bretschneideri in all surveyed provinces except in Shandong, where C. siamense was dominant. In contrast, only C. siamense and C. fioriniae were isolated from P. communis, with the former being dominant. In order to prove Koch's postulates, pathogenicity tests on pear leaves and fruits revealed a broad diversity in pathogenicity and aggressiveness among the species and isolates, of which C. citricola, C. jinshuiense, C. pyrifoliae, and C. conoides appeared to be organ-specific on either leaves or fruits. This study also represents the first reports of C. citricola, C. conoides, C. karstii, C. plurivorum, C. siamense, and C. wuxiense causing anthracnose on pear.

Jugular arginine infusion relieves lipopolysaccharide-triggered inflammatory stress and improves immunity status of lactating dairy cows.

The objective of this study was to evaluate the effects of jugular l-Arg infusion on performance and immune function during lipopolysaccharide (LPS)-induced inflammation of lactating dairy cows. Eight Holstein cows (multiparous, 608.8 ± 31.5 kg) at mid-lactation were randomly assigned to 5-d jugular infusions of control (saline), Arg (3 g/h), LPS (0.033 µg/kg per h), and LPS + Arg (0.033 µg/kg per h of LPS and 3 g/h of Arg) in a replicated 4 × 4 Latin square design with 4 infusion periods separated by 10-d noninfusion periods. Jugular solutions of saline, Arg, LPS, and LPS + Arg were continuously infused using peristaltic pumps for approximately 6 h/d during infusion periods. Milk yield was measured on each day of the infusion period. Milk samples were obtained on the last 2 d of each infusion period, and blood samples were obtained on the last day of each infusion period before infusion (0 h) and at 3 and 6 h. We found that the jugular LPS infusion significantly increased serum concentrations of IL-1ß, IL-6, tumor necrosis factor, inducible nitric oxide synthase, and lipopolysaccharide binding protein, whereas Arg attenuated the increase in IL-6 and inducible nitric oxide synthase levels and tended to decrease the lipopolysaccharide binding protein level. Arginine alleviated the decrease in dry matter intake and milk fat yield and the increase of somatic cell count induced by LPS. Total casein in milk was decreased during the LPS-induced inflammation period, and jugular Arg infusion significantly increased the content of total casein. In contrast, lactalbumin in milk increased during the LPS-induced inflammation period, whereas jugular Arg infusion significantly decreased the content of lactalbumin. The concentrations of plasma Gly, Thr, Ile, Leu, Arg, Phe, and total free AA were significantly decreased by LPS treatment, but Arg attenuated this tendency. These results indicated that jugular Arg infusion (18 g/d) has protective effects on relieving inflammatory stress and improving immunity status triggered by LPS. In conclusion, Arg could attenuate inflammatory stress and improve milk performance of lactating dairy cows. This protective effect may be due to the ability of Arg to suppress LPS effects and improve immunity status.

SNPs detected in the yak MC4R gene and their association with growth traits.

MC4R (melanocortin 4 receptor) is expressed in the appetite-regulating areas of the brain and takes part in leptin signaling pathways. Sequencing of the coding region of the MC4R gene for 354 yaks identified the following five single nucleotide polymorphisms (SNPs): SNP1 (273C>T), SNP2 (321 G>T), SNP3 (864 C>A), SNP4 (1069G>C) and SNP5 (1206 G>C). SNP1, SNP2 and SNP3 were synonymous mutations, whereas SNP4 and SNP5 were missense mutations resulting in amino acid substitutions (V286L and R331S). Pairwise linkage disequilibrium (LD) analysis indicated that two pairs of SNPs, SNP2 and SNP5 (r(2)=0.81027) and SNP4 and SNP5 (r(2)=0.53816), exhibited higher degrees of LD. CC genotype of SNP4, CGACG and CTCCC haplotypes for all SNPs were associated with increased BW of animals that were 18 months old and with the average daily gain. The secondary structure and transmembrane region prediction of the yak MC4R protein suggested that SNP4 was correlated with influential changes in the seventh transmembrane domain of the MC4R protein and with the functional deterioration or even incapacitation of MC4R, which may contribute to the increased feed intake, BW and average daily gain of the yaks with CC genotypes. The data from this study suggested that 1069G>C SNP of the MC4R gene could be used in marker-assisted selection of growth traits in the Maiwa yak breed.

First Report of Chilli veinal mottle virus Infecting Tomato (Solanum lycopersicum) in China.

Chilli veinal mottle virus (ChiVMV), a potyvirus, is widespread over the world. In China, it was first reported in chili pepper (Capsicum annuum) in Hainan Province (south China) in 2006 (2). Subsequently, it was reported in tobacco (Nicotiana tabacum) in Yunnan Province (southwest China) in 2011 (1). Sichuan Province is one of the largest vegetable producing areas of China. In May 2012, tomatoes with leaves displaying virus-infected symptoms like mottling, mosaic, narrowing, or curling were observed in several fields of Chengdu, eastern Sichuan Province, southwest China. Of the 20 fields we investigated, four fields with 90% tomato plants were infected. During 2012 and 2013, six samples were collected from symptomatic tomato leaves based on different symptoms and locations. All six samples were assayed by western blotting using polyclonal antisera (Cucumber mosaic virus [CMV], Tobacco mosaic virus [TMV]) obtained from Agdia (Elkhart) and one antiserum to ChiVMV obtained from Yunnan Academy of Agricultural Science (China). Two samples from Pengzhou and one sample from Shuangliu exhibiting mosaic leaves were positive for TMV, one sample from Pixian exhibiting narrowing leaves was positive for CMV, and the other two samples from Shuangliu exhibiting mottle and leaf distortion were positive for ChiVMV. Total RNAs was extracted from all six samples and healthy tomato leaves using Trizol reagent (Invitrogen), First-strand cDNA synthesis primed with oligo(dT) by SuperScript III Reverse Transcriptase (Invitrogen). RT-PCR was performed using primer pairs ChiVMV-CP F (5'-GCAGGAGAGAGTGTTGATGCTG-3') and ChiVMV-CP R (5'-(T)16AACGCCAACTATTG-3'), which were designed to direct the amplification of the entire capsid protein (CP) gene and 3' untranslated region (3'-UTR) of ChiVMV (GenBank Accession No. KC711055). The expected 1,166-bp DNA fragment was amplified from the two tomato samples from Shuangliu that were positive for ChiVMV in the western blot tests, but not from the others. The obtained fragments were purified and cloned into the PMD18-T vector (TaKaRa) and sequenced. The sequencing results showed that the two ChiVMV isolates from tomato in Shuangliu were identical (KF738253). Nucleotide BLAST analysis revealed that this ChiVMV isolate shared ~84 to 99% nucleotide identities with other ChiVMV isolates available in GenBank (KC711055 to KF220408). To fulfill Koch's postulates, we isolated this virus by three cycle single lesion isolation in N. tabacum, and mechanically inoculated it onto tomato leaves. The same mottle and leaf distortion symptoms in systemic leaves were observed. Subsequent RT-PCR, fragment clone, and sequence determination tests were repeated and the results were the same. All the evidence from these tests revealed that the two tomato plants were infected by ChiVMV. To our knowledge, this is the first report of ChiVMV naturally infecting tomato in China. It shows that ChiVMV is spreading in China and is naturally infecting a new solanaceous crop in the southwest area, and the spread of the virus may affect tomato crop yields in China. Thus, it is very important to seek an effective way to control this virus. References: (1) M. Ding et al. Plant Dis. 95:357, 2011. (2) J. Wang et al. Plant Dis. 90:377, 2006.