Chinese Academy Of Agricultural Sciences Research Articles

Phenotypic evaluation of the Chinese mini-mini core collection of peanut (Arachis hypogaea L.) and assessment for resistance to bacterial wilt disease caused by Ralstonia solanacearum

In order to utilize germplasm resources more efficiently for peanut (Arachis hypogaea L.) genetic improvement, a core collection of 576 accessions and a primary mini core collection of 298 accessions were developed previously from a collection of 6839 cultivated peanut lines stored at the Oil Crops Research Institute of Chinese Academy of Agricultural Sciences at Wuhan. For an efficient evaluation and characterization of the most useful agronomic and disease-resistant traits, an even smaller collection of peanut accessions that represent a spectrum of phenotypes could be more desirable. For this reason, a mini-mini core collection with 99 accessions from the core accessions was developed based on the analysis of 21 morphological traits. It was demonstrated that there were no significant differences between the core and mini-mini core collections in 20 out of the 21 morphological traits studied. Further, the mini-mini core collection captured the ranges of all of the 21 traits displayed in the core collection. The newly developed mini-mini core collection was assessed for resistance to bacterial wilt disease caused by Ralstonia solanacearum. Two accessions showing a high level of resistance to bacterial wilt were identified, demonstrating the usefulness of the mini-mini core collection. The mini-mini-core collection provides a more efficient means of germplasm evaluation and will be resequenced as part of the International Peanut Genome Consortium sequencing project at the UC-Davis Genome Center.

Effort and Contribution of T‐DNA Insertion Mutant Library for Rice Functional Genomics Research in China: Review and Perspective

With the completion of the rice (Oryza sativa L.) genome-sequencing project, the rice research community proposed to characterize the function of every predicted gene in rice by 2020. One of the most effective and high-throughput strategies for studying gene function is to employ genetic mutations induced by insertion elements such as T-DNA or transposons. Since 1999, with support from the Ministry of Science and Technology of China for Rice Functional Genomics Programs, large-scale T-DNA insertion mutant populations have been generated in Huazhong Agricultural University, the Chinese Academy of Sciences and the Chinese Academy of Agricultural Sciences. Currently, a total of 372,346 mutant lines have been generated, and 58,226 T-DNA or Tos17 flanking sequence tags have been isolated. Using these mutant resources, more than 40 genes with potential applications in rice breeding have already been identified. These include genes involved in biotic or abiotic stress responses, nutrient metabolism, pollen development, and plant architecture. The functional analysis of these genes will not only deepen our understanding of the fundamental biological questions in rice, but will also offer valuable gene resources for developing Green Super Rice that is high-yielding with few inputs even under the poor growth conditions of many regions of Africa and Asia.

Gibel carp Carassius auratus gut microbiota after oral administration of trimethoprim/ sulfamethoxazole

Trimethoprim/sulfamethoxazole is widely used in the treatment of infectious diseases caused by bacterial pathogens in aquaculture. However, the practice of antibiotic administration can promote the emergence of resistant strains of bacteria and result in a wane in efficacy over time. The objective of this study was to assess the effect of oral treatment with trimethoprim/sulfamethoxazole on the gastrointestinal (GI) microbiota of healthy gibel carp and those affected with bacterial enteritis. By using denaturing gradient gel electrophoresis (DGGE), the changes in the predominant bacterial communities were directly depicted for the first time. The main findings were (1) Actinobacteria, Firmicutes and Proteobacteria were the predominant phyla in the healthy gibel carp intestine; (2) administration of antibiotics had a more profound impact on the intestinal microflora of healthy fish than of the diseased ones; and (3) Enterobacteriaceae might be one of the major drug-resistant bacteria in the gibel carp intestine. This study provides an insight into the effect of antibiotic treatment on the establishment and colonization of fish GI microbiota and speculates on some possible drug-resistant bacteria.

Development of Industrialization Process of Pesticide Residue Analysis Grade <i>n</i>-Hexane

Pesticide residue grade solvents have the characteristic of ultra high purity, low UV value, low pH value, low moisture content and low evaporation residue, which do not have those controlled impurity peak in pesticide chromatographic detection. This project is funded by the China government to develop the industrialization process of pesticide residue grade n-hexane, in which the industrial n-Hexane is used as crude purified by decoloration, distillation and filtration process. The products are validated by National Research Center for Environmental Analysis and Measurement (CNEAC), National Research Center for Geoanalysis, Chinese Academy of Inspection and Quarantine (CAIQ), Chinese Academy of Agricultural Sciences (CAAS) and other government originations for polychlorinated biphenyls, organochlorine pesticide or chiral pesticides analysis and further confirmed that it’s competitive to all others imported n-Hexane currently occupied in China. The optimized process parameters are: firstly utilizing 2% of silicon dioxide as decoloration agent then rinsed with distillation water before distillation. The heating temperature of two distillation towers is 88°C with reflux ratio of 15:1 and 4:1 respectively, the discharge rate of heavy component is 20 mL/min, the yield of this process is approximately 70%. This patented technique will meet pesticide residue grade n-Hexane market in China and seek for cooperation globally.

Current status of the giant freshwater prawn (Macrobrachium rosenbergii) industry in China, with special reference to live transportation

The giant freshwater prawn Macrobrachium rosenbergii was first introduced into mainland China by the Chinese Academy of Agricultural Sciences from Japan in 1976. It was cultured at the Guangdong Province Fishery Institute (now as Pearl River Fishery Institute, Chinese Academy of Fishery Sciences). Following these early efforts, giant freshwater prawn culture has gradually spread to northern and inland areas. At this time (2011), China has become the largest producer of giant freshwater prawn in the world. Due to a preference of cooking with fresh aquatic products, Chinese retail markets have developed which have special live-holding counters. From small rural shops to urban supermarkets and specialty restaurants, live seafoods are commonly available whereby customers pick out the products they wish to purchase and consume. Giant freshwater prawns are produced in over 35 countries throughout the world. They are usually sold either fresh, on-ice, or frozen. What makes the Chinese prawn industry unique is its focus on a live transportation and marketing model. As a result, numerous innovative live-transport technologies have been developed which have been key to the success of this expanding domestic industry. In this article, we would like to give a general overview of the giant freshwater prawn industry, its status in China, and the special role live-transportation has for expanding production of giant freshwater prawns.

The Application of Hyper-spectral Data and RBF Neural Network Method to Retrieval of Leaf Area Index of Grassland

In accordance with the 120 sites of grassland canopy spectral reflectance and the leaf area index(LAI) data collected by Chinese Academy of Agricultural Science,the method of Radial Basis Function(RBF) neural network was developed for the prediction of LAI after the compression of spectral reflectance using principal component analysis(PCA).The PCA results show that the cumulative reliability of the first 9 PCs is up to 99.782%,covering the majority of original spectral information.The 120 sites of LAI and 9 PC samples were classified randomly for training dataset(90 sites) and predicting dataset(30 sites),and were used to establish the neural network and predict the LAI,respectively.The results show that the accuracy rate of training data is up to 100%(RMSE=0.009 6,R2=0.999).The root mean square error(RMSE) and correlation coefficient(R2) for the prediction dataset are 0.839 and 0.218 6 respectivdg,thus achieving more preferable results and improved the accuracy(RMSE=0.416 5,R2=0.570)of the traditional multiple linear regression method.

New Technologies, Tools and Approaches for Improving Crop Breeding

New Technologies, Tools and Approaches for Improving Crop Breeding

Molecular breeding in plants: moving into the mainstream

Molecular breeding, including both marker-assisted breeding and genetic engineering, has experienced significant innovations and advances during the past three decades. Starting with Mendelian genetics, plant breeders have progressed from using morphological markers to protein isozymes and then ultimately to DNA-based markers, which have enabled routine genome-wide analysis. As a result, thousands of genes and quantitative trait loci (QTL) have been mapped across the major crop species, laying the foundation for marker-assisted selection (MAS) techniques, such as precise marker-assisted backcrossing, to transfer desirable loci into breeding lines. With the subsequent development of advanced MAS strategies, including QTL pyramiding, marker-assisted recurrent selection and genomic selection, marker-assisted approaches have led to rapid gains in selection for plant breeding programs. Breeding by design and genetic modeling have received great attention as designing a desirable plant based on marker and associated gene information becomes increasingly possible. Following rice as the first crop species sequenced, whole genome sequences have been increasingly available for more and more crop species, enabling genotyping by sequencing and sequence-based marker technology and molecular breeding strategies to become a viable option, one widely used in large multinational seed companies. With significant reduction of genotyping cost and increased throughput, next-generation sequencing and SNP genotyping technologies are increasingly making MAS a choice of public-sector plant breeding as well. Now crop genomic diversity can be characterized at the sequence level based on haplotype maps, and molecular breeding can be further accelerated through allele mining and haplotype-based design and selection. With all the developments and significant advances in various fields of molecular biology and their integration with platforms and tools, molecular breeding is rapidly moving into the vanguard of mainstream breeding programs. To review and evaluate new theories and technologies recently developed in relation to plant breeding, the 3rd International Conference on Plant Molecular Breeding (ICPMB) was held in Beijing, China, Sept. 5–9, 2010. The conference was organized by an International Organization Committee represented by Zhi-Kang Li (Chinese Academy of Agricultural Sciences/International Rice Research Institute), Jean-Marcel Ribaut (the Generation Challenge Program), Aimin Zhang (Chinese Y. Xu (&) Institute of Crop Sciences/International Maize and Wheat Improvement Center (CIMMYT), Chinese Academy of Agricultural Sciences, 100081 Beijing, China e-mail: y.xu@CGIAR.ORG

Industrialization Process of Pesticide Residue Grade n-Hexane

This project is funded by the China government to develop the industrialization process of pesticide residue grade n-hexane, in which the industrial n-Hexane is used as crude purified by decoloration, distillation and filtration process. The products are validated by National Research Center for Environmental Analysis and Measurement (CNEAC), National Research Center for Geoanalysis, Chinese Academy of Inspection and Quarantine (CAIQ), Chinese Academy of Agricultural Sciences (CAAS) and other government originations for polychlorinated biphenyls, organochlorine pesticide or chiral pesticides analysis and further confirmed that it’s competitive to all others imported n-Hexane currently occupied in China. This patented technique will meet pesticide residue grade n-Hexane market in China and seek for cooperation globally.

施加角担子菌B6对连作西瓜土壤微环境和西瓜生长的影响

在盆栽条件下,研究了施加角担子菌B6的菌丝对连作西瓜的土壤微生物区系以及产量的影响,以探索西瓜连作障碍的生物防治措施。施加B6的活菌丝(C)显著减少土壤中真菌的数量、增加细菌/放线菌的比例,在西瓜成熟期,与对照(A)和施加灭活的B6菌丝(B)相比,土壤中尖孢镰刀菌(FO)的数量分别减少了29.9%和63.3%。相比对照(A),在成熟期,C处理中土壤脲酶、蔗糖酶和多酚氧化酶的活力分别提高了19.0%、159.0%和31.3%;西瓜超氧化物岐化酶(SOD)和过氧化物酶(POD)活性分别增加32.7%和4.6%,西瓜根系活力(TTC法)增强46.2%,丙二醛(MDA)含量减少51.4%。与对照(A)和施加灭活B6菌(B)相比,施加B6菌(C)后,西瓜单果重分别增加44.8%和40.9%,总产量分别增加103.8%和64.9%,可溶性糖含量分别增加35.1%和10.0%。施加B6的活菌丝能够通过改善土壤微环境,提高西瓜植株的抵抗力,进而增加产量。;The deterioration of the soil microbial population and the decline of enzyme activity have been regarded as two key factors in the reduction of watermelon yield in long-term continual watermelon cropping systems. To replicate the watermelon cropping system as much as possible, a surface layer (50-200 mm depth) of an Ultisol was sampled from a 3 year-old continuously cropped upland watermelon site, at the Red Soil Experimental Station, Chinese Academy of Agricultural Sciences, Yongzhou, Hunan Province (N26°45',E111°53'). Pot experiments were conducted to investigate the effects of the addition of <em>Ceratobasidium stevensii</em> B6 on watermelon (<em>Citrullus lanatus</em>) yield (and the associated soil microflora) to study biological control measures for a long-term continual watermelon cropping system. Three treatments were used a control (A), an inactivated B6 mycelia (B) and an activated B6 mycelia treatment (C). At the harvest stage, the application of living B6 mycelia (Treatment C) significantly reduced the number of soil fungal colony forming units (CFU) by 17%, but the quantity of bacteria and actinomycetes increased by 2.2 and 0.4 fold respectively, compared with the control (A). For the treatment using the activated B6 mycelia (C), the quantity of <em>Fusarium oxysporum</em> (FO) in the soil at the harvest stage decreased by 29.9% and 63.3% compared with the control (A) and the inactivated B6 mycelia (B) respectively. Meanwhile, the activities of soil urease, sucrase and polyphenol oxidase were enhanced by 19.0%, 159.0% and 31.3% in treatment C compared with treatment A, respectively. Moreover, the activities of superoxide dismutase and peroxidase in the watermelon leaves also increased by 32.7% and 4.6% when compared with treatment A, respectively. In other words, the activated B6 mycelia significantly induced a systemic resistance of watermelons to the invasion of FO. Furthermore, in contrast with treatment A, root activities in treatment C were increased by 46.2% while the malonaldehyde content (MDA) in the leaves was reduced by 51.4%. Compared with the control (A) and inactivated B6 mycelia (B), the individual fruit weight increased by 44.8% and 40.9% respectively in the treatment where activated B6 mycelia were added. Total yield was also increased by 103.8% and 64.9%, respectively. The soluble sugar content of the fruit likewise was increased by 35.1% and 10.0% for treatment C when compared with the other treatments. Although the differences in the vitamin C concentration of the watermelon fruit between all the treatments was not significant, the quality of the watermelons in treatment C was improved, as evidenced by an increase in the soluble sugar content. These results suggested that the addition of the activated B6 mycelia for improving the soil microenvironment and to increase the watermelon yield has promise, and may be able to be commercially used in the future; hence, further research is required on the mechanisms involved.

Methodology A new strategy employed for identification of sweet orange cultivars with RAPD markers

We optimized RAPD techniques by increasing the length of RAPD primers and performing a strict screening of PCR annealing temperature to distinguish 60 sweet orange cultivars from the Research Institute of Pomology at the Chinese Academy of Agricultural Sciences. A new approach called cultivar identification diagram (CID) was used to improve the efficiency of RAPD markers for cultivar identification. Thirteen effective primers were first screened from 54 RAPD arbitrary 11-mer primers based on their amplification products and amplified polymorphic bands; they were then used for PCR amplification of all 60 cultivars. All cultivars were manually and completely separated by the polymorphic bands appearing in DNA fingerprints from 13 primers; a CID of the 60 sweet orange cultivars was then constructed. This CID separated all the cultivars from each other, based on the polymorphic bands; the corresponding primers were marked in the correct positions on the sweet orange CID. The CID strategy facilitates the identification of fruit cultivars with DNA markers. This CID of sweet orange cultivars will be very useful for the protection of cultivar rights and for early identification of seedlings in the nursery industry.

Genetic Effects of Background‐Independent Loci for Grain Weight and Shape Identified using Advanced Reciprocal Introgression Lines from Lemont × Teqing in Rice

ABSTRACTGenetic background effect and environmental sensitivity are two major obstacles for marker‐assisted breeding (MAB) of complex traits. Background‐independent (BI) loci for 1000‐grain weight (TGW) and grain shape (GS) were detected over three seasons using a set of reciprocal introgression lines (ILs) from Lemont × Teqing cross with parents having distinctive GS but equal TGW. Reciprocal ILs in advanced (F11–13) generations were genotyped using 142 polymorphic simple sequence repeat (SSR) markers on 64 chromosome bins. Nineteen bins of BI loci significantly associated with TGW and/or GS that were identified consistently over multiple seasons and additive effects were in the opposite directions at the same loci between two backgrounds. These 19 bins could be classified into three types: Type I includes four genetic overlapping bins affecting both TGW and GS, Type II includes nine genetic overlapping bins controlling GS component traits, and Type III includes six bins for single GS component. Among them, five stably expressed BI loci (bins 2.2, 2.5, and 3.7 for grain length [GL] and bins 5.2 and 7.4 for grain width [GW]) found consistently over three seasons would be the first choice for MAB. Secondary choice would be the loci that expressed over at least over two seasons and include bins 1.2, 3.3, 4.3 for GL and bins 7.1 and 11.5 for GW. Two related key traits, one for yield (TGW) and the other for grain quality (GS), can now be fully utilized through marker‐aided transfer or pyramiding of the identified BI loci in rice (Oryza sativa L.) improvement programs.

Comparison of quantitative trait loci for rice yield, panicle length and spikelet density across three connected populations

1National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China 2College of Biosafety Science and Technology Hunan Agricultural University, Changsha 410128, People’s Republic of China 3Present address: Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, People’s Republic of China

Genomics of plant genetic resources: an introduction

This special issue of Plant Genetic Resources has assembled 52 short articles selected among the over 350 oral and poster communications presented during the 2nd International Symposium on Genomics of Plant Genetic Resources (GPGR2; www.GPGR2.com) held in Bologna, Italy, from April 24 to 27, 2010. The second edition of GPGR2, co-organized by Bioversity International, the Leibniz Institute of Plant Genetics and Crop Plant Research and the University of Bologna, followed the first edition organized in 2005 by the Chinese Academy of Agricultural Science in Beijing, China...

Industry Watch

MDI and Integrated Sciences Enter Exclusive Distribution Agreement in Australia. Agenix, IMB to Develop Hep B Drug in China. NeoStem to Acquire Progenitor Cell Therapy in $20m Deal. Origin Biotechnology Reaches Worldwide Agreement with Chinese Academy of Agricultural Sciences for Bt-gene. Transasia Bio-Medicals Acquires Pliva Lachema Diagnostika. Advinus Team Discovers Novel Molecule for Treatment of Diabetes. Biocon Partners with Teleradiology Solutions. Abbott, Reata Pharma Collaborate on Kidney Treatment.

First Detection of Virulence in Puccinia striiformis f. sp. tritici in China to Resistance Genes Yr24 (=Yr26) Present in Wheat Cultivar Chuanmai 42.

Stripe rust disease of wheat caused by Puccinia striiformis f. sp. tritici was observed on previously resistant bread wheat (Triticum aestivum L.) cv. Chuanmai 42 during the 2008-2009 crop season in Pi County, Sichuan Province, China. More than 10 single pustules were isolated from the diseased leaf samples collected in the field and inoculated on 7-day-old susceptible wheat seedlings cv. Mingxian 169. After 18 to 24 h of incubation at 100% relative humidity in darkness, the plants were moved into the greenhouse, maintained at 15 to 18°C, and supplemented with 10,000 lx of fluorescent light for 10 h per day. The second leaves were clipped when chlorotic spots appeared on leaves (~7 days postinoculation), and plants were covered with glass cylinders to prevent cross contamination. Urediniospores of each isolate were collected 16 days after inoculation and temporarily kept in a dryer at low temperature (3 to 4°C). The virulence spectra of the isolates were tested on Chinese differentials and wheat lines with known Yr genes at the seedling stage (1). A new Yr24 (=Yr26) virulent pathotype, different from currently known pathotypes in China, was identified. To our knowledge, this is the first detection of Yr24 virulence in Puccinia striiformis f. sp. tritici populations on Chuanmai 42. In addition, the new pathotype was also virulent to Lantian 17, Guinong 22 (Chinese differential), and 92R137 derived wheat lines Nannong 04Y10 and Nannong 05Y628, known to carry Yr24 (2,3). The avirulence/virulence formula of the new pathotype is Yr1, 3, 4, H46, 5, 6, 15, 17, 18, 32, Sp, Sd/Yr2, 8, 9, 10, 12, 24 (=26), 31, and Su. Wheat cultivars and breeding materials, previously protected by Yr24 gene, are now vulnerable to stripe rust epidemics in the region. Pure isolates of the new pathotype (Accession No. 09-6-16-3) are stored in the Chinese Academy of Agricultural Sciences (CAAS; Beijing) stripe rust collection.

Bivalent Insect-Resistant Gene Transgenic Cotton Variety CCRI 41 with High Efficiency and Broad Aadaptability

Bollwarm has been a serious problem in cotton production in China since the 1990th. To breed transgenic pest-resistant cotton variety is one of the most economic measures. Bivalent insect-resistant Bt + CpTI gene transgenic cotton variety possesses stronger pest resistance compared with Bt cotton. CCRI 41 is an insect-resistant gene transgenic cotton variety bred by Cotton Research Institute, Chinese Academy of Agricultural Sciences. A bivalent insect-resistant Bt+CpTI gene was transformed into a high-performance conventional cotton variety, and stable and high-performance transgenic lines were selected using stress-resistance screening and molecular assistant selection strategies. It was the first national released bivalent insect-resistant transgenic cotton variety in China. The cumulative extension acreage of CCRI 41 has reached 3 million ha extensive popularization of CCRI 41 since 2002 in 6 provinces in Yellow River Valley and has reaped economic benefits of 4.5 billion RMB yuan in total. These scientific achievements won the 2009 State Science and Technology Awards of China (2nd prize).

Origin Analysis of Resistance Gene to Soybean Mosaic Virus in Soybean Line ICGR95-5117

Soybean mosaic virus (SMV) is a prevalent viral pathogen of soybean. The soybean mosaic virus N3 (SMV3) is the most toxic strain in northeast China. Soybean lines ICGR95-5117 and ICGR95-5383 with high resistance to SMV3,which were all derived from ICGR661,are recently released from Chinese Academy of Agricultural Sciences. Several genes resistant to soybean mosaic virus have been identified and mapped on different soybean linkage groups by molecular markers. The re-sistance gene in ICGR95-5383 has been mapped on soybean linkage group F near Rsv1 gene. A dominant marker developed from the candidate gene of Rsv1 has been reported recently. The purpose of this research was to find the origin of the SMV3 resistance gene in ICGR95-5117 and the relationship of this resistance gene with Rsv1 by analyzing the pedigree. Thirty four SSR markers selected from linkage group F and two markers,SCAR marker (SCN11) tightly linked with resistance gene in ICGR95-5383 and Rsv1-f/r makers co-segregated with resistance gene Rsv1,were used to detect the materials in pedigree of ICGR95-5117. The similarity coefficient of the two lines between ICGR95-5117,ICGR95-5383 and ICGR661 was the highest. All the material except for Mangnolid(F-53)B had SMV3 resistance allele at locus SCN11. Williams 82 and ICGR661 had the same allelic variation at Rsv1-f/r locus with ICGR95-5117,revealing that the Rsv1 gene in ICGR95-5117 was inherited from Williams 82 via ICGR661. But ICGR95-5117 had higher resistance than ICGR661,indicating it might inherit different genes from SMV3 resistance parent Ludou4.

First Report of Grapevine leafroll-associated virus 4 and 5 in Grapevines in China.

Grapevine leafroll disease (GLD) is one of the most important diseases of grapevines worldwide. Nine serologically distinct viruses in the Closteroviridae family are associated with GLD. Previous studies reported that Grapevine leafroll-associated virus (GLRaV) -1, -2, -3, and -7 were present in grapevines in China with GLRaV-1 and -3 the predominant viruses associated with GLD (1). To confirm if GLRaV-4 and -5 were also present in China, 36 dormant canes from individual vines of 29 cultivars that showed GLD leaf symptoms during the growing season were collected from the germplasm collection plot of the Research Institute of Pomology, Chinese Academy of Agricultural Sciences. Total RNA extracted by a silica capture protocol (2) from phloem-enriched bark of 36 samples was tested separately for GLRaV-4 and -5 by reverse transcription (RT)-PCR using virus-specific primers. Primers LR4F (5'-ACATTCTCCACCTTGTGCTTTT-3') and LR4R (5'-CATACAAGCGAGTGCAATTAC-3') (4) were used to amplify a 321-bp fragment corresponding to a partial region of the HSP70 gene from GLRaV-4. One sample from cv. Autumn Royal was infected by GLRaV-4. The amplicon was cloned and a single clone was sequenced (GenBank Accession No. GQ246624) that showed 99% nucleotide identity with a corresponding region of a GLRaV-4 isolate from the United States (Accession No. AF039553). Since antiserum specific to GLRaV-4 was unavailable, a second pair of primers, LR4CP-F (5'-GGTGTCCAGCGCTTCCAA-3') and LR4CP-R (5'-GCCAGAGAAGCATCGTAA-3'), was designed on the basis of the sequence of GLRaV-4 from Chile (Accession No. EU746620) that amplified a 300-bp fragment specific to the coat protein gene of GLRaV-4. The amplicon was cloned and a single sequence (Accession No. GQ479041) was compared with a corresponding nucleotide sequence of GLRaV-4 from Chile (Accession No. EU746621) showing 99% identity. A sample from cv. Malaga Rose was positive when tested by ELISA with antibodies specific to GLRaV-5 (Neogen Europe, Ltd. Scotland, UK) and this was confirmed by amplification of a 690-bp fragment corresponding to the GLRaV-5 coat protein gene using virus-specific primers LR5F (5'-CCCGTGATACAAGGTAGGACA-3') and LR5R (5'-CAGACTTCACCTCCTGTTAC-3') (3). The amplicon was cloned and a single clone was sequenced (Accession No. GQ246625) that showed 95% nucleotide identity with the CP gene sequence of GLRaV-5 from Argentina (Accession No. EU815935). To our knowledge, this is the first report of GLRaV-4 and -5 in grapevines in China. Confirmation of these viruses in China is very important for producing virus-free plants and this information also will be helpful in developing a multiplex RT-PCR assay to simultaneously detect multiple GLRaVs and helpful with studies on the molecular variability of these viruses.

Microcalorimetric assessment of microbial activity in long-term fertilization experimental soils of Southern China

Microcalorimetry, plate count and PCR-denaturing gradient gel electrophoresis (DGGE) were employed to investigate microbial diversity and activity in soils from the Red Soil Experimental Station of the Chinese Academy of Agricultural Sciences, Hunan Province, China, where a wheat-corn rotation with 12 fertilization treatments was established in 1990. Fertilization greatly increased microbial biomass carbon (C) and nitrogen (N) (C(mic) and N(mic)) as well as the activities of phosphatase, urease, invertase, protease, catalase and dehydrogenase. Manure alone (M) enhanced the number of denitrifying and aerobic bacteria by 54.4% and 20.5%, respectively, whereas fallow (H) increased the number of aerobic cellulose decomposing bacteria by 31.4%. Fallow and soils amended with mineral fertilizers plus pig manure or straw increased both the DGGE band patterns and the Shannon index compared with mineral fertilizers or the control. Mineral treatments with lower bacterial numbers enhanced the values of the peak time (t(max)) more than did organic treatments. The peak height (P(max)) was positively correlated (P<0.01), with soil enzymes, C(mic) and N(mic), and the number of microorganisms, whereas the peak time (t(max)) was negatively connected (P<0.01) with these parameters. The microbial growth rate constant (k) was linked to bacteria (P<0.01), actinomycetes (P<0.05) and catalase (P<0.05). The total heat evolution (Q) had no relationships with any soil microbial properties (except for catalase). We propose that P(max) and t(max) could be used as indices of soil microbial activity, while the values of k and Q are poor indicators.