Preliminary Studies on Species and Distribution of Citrus Viroids in China

Citrus viroids are the small but economically important RNA pathogens. For investigating their occurrence and distribution in China, 65 viroid samples collected from 8 major citrus cultivated regions were evaluated using one-step or multiplex onestep RT-PCR and biological indexing for specifically detection of Citrus exocortis viroid （CEVd）, Citrus bent leaf viroid （CBLVd）, Hop stunt viroid （HSVd）, Citrus viroid-Ⅲ （CVd-Ⅲ） and Citrus viroid-Ⅳ （CVd-Ⅳ）. The results showed that there were at least 4 kinds of citrus viroids （CEVd, CBLVd, HSVd, and CVd-Ⅲ） on citrus trees in China. Most of the infected citrus plants harbored more than one viroid species, and two plants were infected with up to 4 citrus viroids. Sweet orange was more frequently infected by viroids than other citrus varieties. It is the preliminary report on the species and distribution of citrus viroids in China.     

Genetic Evolution Analysis on Wild Isolates of Citrus Tristeza Virus Originated in China Based on Coat Protein Genes Sequences

The coat protein （CP） genes were cloned and sequenced from viral particles of 11 isolates of citrus tristeza virus （CTV） collected from wild citrus plants in China and 4 Chinese isolates from cultivated sweet orange and pummelo varieties, respectively. By analyzing and comparing the nucleotide and amino acid sequences of CP genes, the 11 wild CTV isolates were found over 92% identical with 4 Chinese CTV isolates and 21 exotic CTV isolates from cultivated citrus. From 91 to 100% of the CTV CP gene sequences in wild type citrus plants were generally well conserved. Genetic evolution analysis indicated that the GC% of the CP gene was less than AT%, and more transition were found in the CP genes than transversion with the transition/transversion ratio ranging from 6.3 to 7.0 among species. The substitution frequency was the highest at the third codon, followed by the first and second codon. The ratio of non-synonymous mutations （du） to synonymous mutations （ds） was far lower than 1, suggesting that the CP gene might have experienced purifying selection in the evolution. Phylogenetic analysis revealed that the 11 CTV isolates in Chinese wild type citrus belonged to different phylogenetic clusters, and shared higher homology and closer relationships with other cultivated citrus CTV isolates from different countries, which indicated complicated genetic relationships among the CTV isolates. In addition, CTV isolates with similar biological characteristics usually located into the same clusters. Therefore, the conclusion was drawn that pathogenicity was critical to evolution and origin of CTV.     

Effect of nitrogen fertilization on yield,N content,and nitrogen fixation of alfalfa and smooth bromegrass grown alone or in mixture in greenhouse pots

Planting grass and legume mixtures on improved grasslands has the potential advantage of realizing both higher yields and lower environmental pollution by optimizing the balance between applied N fertilizer and the natural process of legume biological nitrogen fixation. However, the optimal level of N fertilization for grass-legume mixtures, to obtain the highest yield, quality, and contribution of N2 fixation, varies with species. A greenhouse pot experiment was conducted to study the temporal dynamics of N2 fixation of alfalfa(Medicago sativa L.) grown alone and in mixture with smooth bromegrass(Bromus inermis Leyss.) in response to the addition of fertilizer N. Three levels of N(0, 75, and 150 kg ha–1) were examined using 15N-labeled urea to evaluate N2 fixation via the 15 N isotope dilution method. Treatments were designated N0(0.001 g per pot), N75(1.07 g per pot) and N150(2.14 g per pot). Alfalfa grown alone did not benefit from the addition of fertilizer N; dry matter was not significantly increased. In contrast, dry weight and N content of smooth bromegrass grown alone was increased significantly by N application. When grown as a mixture, smooth bromegrass biomass was increased significantly by N application, resulted in a decrease in alfalfa biomass. In addition, individual alfalfa plant dry weight(shoots+roots) was significantly lower in the mixture than when grown alone at all N levels. Smooth bromegrass shoot and root dry weight were significantly higher when grown with alfalfa than when grown alone, regardless of N application level. When grown alone, alfalfa's N2 fixation was reduced with N fertilization(R2=0.9376, P=0.0057). When grown in a mixture with smooth bromegrass, with 75 kg ha–1 of N fertilizer, the percentage of atmospheric N2 fixation contribution to total N in alfalfa(%Ndfa) had a maximum of 84.07 and 83.05% in the 2nd and 3rd harvests, respectively. Total 3-harvest %Ndfa was higher when alfalfa was grown in a mixture than when grown alone(shoots: |t|=3.39, P=0.0096; root: |t|=3.57, P=0.0073). We believe this was due to smooth bromegrass being better able to absorb available soil N(due to its fibrous root system), resulting inlower soil N availability and allowing alfalfa to develop an effective N2 fixing symbiosis prior to the 1st harvest. Once soil N levels were depleted, alfalfa was able to fix N2, resulting in the majority of its tissue N being derived from biological nitrogen fixation(BNF) in the 2nd and 3rd harvests. When grown in a mixture, with added N, alfalfa established an effective symbiosis earlier than when grown alone; in monoculture BNF did not contribute a significant portion of plant N in the N75 and N150 treatments, whereas in the mixture, BNF contributed 17.90 and 16.28% for these treatments respectively. Alfalfa has a higher BNF efficiency when grown in a mixture, initiating BNF earlier, and having higher N2 fixation due to less inhibition by soil-available N. For the greatest N-use-efficiency and sustainable production, grass-legume mixtures are recommended for improving grasslands, using a moderate amount of N fertilizer(75 kg N ha–1) to provide optimum benefits.     

Preliminary Studies on CPG/Hinf Ⅰ RFLP Groups of Citrus tristeza virus Infected Sweet Oranges in China

Citrus tristeza virus （CTV） causes economically important losses to the citrus industry worldwide. Mild strain cross protection （MSCP） against tristeza has hardly been practised due to mixed infection of different CTV-strains and little background of its molecular biology in China. For better cognition on CTV, 192 sweet orange samples collected from eight provinces （Chongqing, Sichuan, Fujian, Hunan, Guangxi, Yunnan, Guangdong and Jiangxi） were tested by direct tissue blot immuno-assay （DTBIA）, and 158 of them were tested positively, which therefore were subjected to coat protein gene （CPG）/Hinf Ⅰ restriction fragment length polymorphism （RFLP） analysis. Sample bulks were compared between Chongqing and Fujian by some statistical data, including ratios of single infection and mixed infection to local samples, proportions of CTV isolates with single RFLP groups, and rates of each RFLP group. The simplified analysis of samples from the other six provinces were then conducted. This study suggests that CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ are the main epidemic ones in China, and mixed infection of CTV in fields are popular. Based on observation of severity of stem-pitting symptom in field trees, CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ caused severe stem-pittings in sweet oranges in China.     

Influence of REV and ALV-J Co-Infection on Immunologic Function of T Lymphocytes and Histopathology in Broiler Chickens

The aim of present investigation is to study the effect of single- and co-infection with REV and ALV-J on T lymphocytes bioactivities and histopathology in broiler chickens. The bioactivities of blood and spleen T lymphocytes including lymphoproliferation responses, cytotoxicitic responses, and histopathology of spleen were detected in broiler chickens singly- or co-infected with REV and ALV-J at different days post inoculation and the virus expressions in spleen of infected broiler chickens were detected with immunofluorescence assay （IFA）. The results indicated that blood and spleen T lymphocytes proliferation responses and cytotoxicity in broilers infected with REV or/and ALV-J were inhibited in the whole observed period compared with controls. In the co-infected chickens they were highly inhibited than in the single-infected. The histopathology of spleen in infected chickens at 17 and 37 d post inoculation （dpi） indicated that cell interium increased, the numbers of lymphocytes decreased, and the regrowth were destroyed or decreased, especially more significantly at 17 than at 37 dpi. The different numbers of virus were detected in spleen lymphocytes in REV- infected and/or ALV-J-infected chickens. In the spleen of co-infected chicken, both REV and ALV-J were detected and the total numbers of viruses were more than in chickens singly-infected with REV or ALV-J. Thus, the co-effect of REV and ALV-J caused more immunosuppression on T lymphocytes bioactivities in broiler chickens than single-effect of ALV-J or REV, which contributed to the sever histopathology and the product of tumor cells. This study will be helpful for understanding the effect of co-infection with many viruses and control them in poultry.     

Transgenic Expression of a Functional Fragment of Harpin Protein Hpa1 in Wheat Represses English Grain Aphid Infestation

The harpin protein Hpa1 produced by the rice bacterial blight pathogen promotes plant growth and induces plant resistance to pathogens and insect pests. The region of 10-42 residues （Hpa110-42） in the Hpa1 sequence is critical as the isolated Hpa110-42 fragment is 1.3-7.5-fold more effective than the full length in inducing plant growth and resistance. Here we report that transgenic expression of Hpa110-42 in wheat induces resistance to English grain aphid, a dominant species of wheat aphids. Hpa110-42-induced resistance is effective to inhibit the aphid behavior in plant preference at the initial colonization stage and repress aphid performances in the reproduction, nymph growth, and instar development on transgenic plants. The resistance characters are correlated with enhanced expression of defense-regulatory genes （EIN2, PP2-A, and GSL10） and consistent with induced expression of defense response genes （Hel, PDF1.2, PR-1b, and PR-2b）. As a result, aphid infestations are alleviated in transgenic plants. The level of Hpa110-42-induced resistance in regard to repression of aphid infestations is equivalent to the effect of chemical control provided by an insecticide. These results suggested that the defensive role of Hpa110-42 can be integrated into breeding germplasm of the agriculturally signiifcant crop with a great potential of the agricultural application.     

Mechanisms involved in biocontrol and plant induced resistance by Trichoderna asperellun (T. harzianum T-203)

Growing awareness of the environmental damage caused by the use of chemical substances for plant disease control in agriculture has raised the need to study biological alternatives, such as activating the defense response of plant crops by inducers not toxic to the environment. Trichoderna spp. are effective biocontrol agents for a number of soilborne pathogens, and are also known for their ability to enhance plant growth and to induce systemic resistance (ISR) in plants.     

Mechanisms involved in biocontrol and plant induced resistance by richoderma. asperellum (T. harzianum T-203)

Growing awareness of the environmental damage caused by the use of chemical substances for plant disease control in agriculture has raised the need to study biological alternatives, such as activating the defense response of plant crops by inducers not toxic to the environment. Trichoderma spp. are effective biocontrol agents for a number of soilborne pathogens, and are also known for their ability to enhance plant growth and to induce systemic resistance (ISR) in plants. In our laborator…     

Changes in Soil Biota Resulting from Growth of the Invasive Weed,Ambrosia artemisiifolia L.（Compositae）, Enhance Its Success and Reduce Growth of Co-Occurring Plants

Exotic plant invasion presents a serious threat to native ecosystem structure and function. Little is known about the role of soil microbial communities in facilitating or resisting the spread of invasive plants into native communities. The purpose of this research is to understand how the invasive annual plant Ambrosia artemisiifolia L. facilitates its competition capacity through changing the structure and function of soil microbial communities. The soil characteristics of different areas invaded by A. artemisiifolia were examined. Greenhouse experiments were designed to assess the effect of A. artemisiifolia invasion-induced changes of soil biota on co-occurring plant growth, and on the interactions between A. artemisiifolia and three co-occurring plant species. The results showed that the soil organic C content was the highest in heavily invaded sites, the lowest in native plant sites, and intermediate in newly invaded sites. Soil available N, P and K concentrations in heavily invaded site were 2.4, 1.9 and 1.7 times higher than those in native plant soil, respectively. Soil pH decreased as A. artemisiifolia invasion intensity increased, and was lower in invaded sites（heavily invaded and newly invaded） than in native plant sites. The soil microbial community structure was clearly separated in the three types of sites, and A. artemisiifolia invasion increased anaerobe, sulfate-reducing bacteria and actinomycete abundance. Soil biota of invaded sites inhibits growth of co-occurring plants（Galinsoga parvifloraCav., Medicago sativa L. and Setaria plicata（Lam.） T. Cooke.） compared to soil biota from un-invaded sites, but facilitates A. artemisiifolia growth and competition with co-occurring plants. A. artemisiifolia biomass was 50-130% greater when competing with three co-occurring plants, compared to single-species competition only（invasion by A. artemisiifolia alone）, in heavily invaded soil. Results of the present study indicated that A. artemisiifolia invasion alters the soil microbial communit     

Effects of INA on postharvest blue and green molds and anthracnose decay in citrus fruit

As a synthetic functional analog of salicylic acid, 2,6-dichloroisonicotinic acid(INA) is effective in inducing the host disease resistance of a plant against a pathogen. The effects of INA on controlling postharvest blue and green molds and anthracnose decay and defense-related enzymes on citrus fruits were investigated, and the ascorbic acid of naturally infected citrus flavedo was also measured. Results showed that 1.0 mmol L~(–1) INA treatments significantly reduced blue and green molds and anthracnose decay development on both wound-inoculated fruit and naturally-infected fruit compared with the control fruit. The treatment effectively enhanced the β-1,3-glucanase(GLU), chitinase(CHI), phenylalanine ammonia-lyase(PAL) and peroxidase(POD) activities and the polyphenol oxidase(PPO) in flavedo. The results presented here suggest that INA might be used as a chemical fungicide substitution to control postharvest diseases in citrus fruits.     

Isolation and Identification of an Endophytic Strain with Antibiosis Ability from Davidia involucrate Brail.

[Objective]The aim was to isolate and identify an endophytic bacteria strain with antimicrobial activity from Davidia involucrate Brail.[Method]Endophytic strain with antibiosis ability was isolated from D.involucrate Brail by using cylinder-plate method.Then,it was identified through physiological and biochemical tests,16S rDNA homology analysis as well as some gene-specific sequence analysis.[Result]B221 stain had antimicrobial activity against a variety of rice plant pathogens,and it was identified as Bacillus subtilis.[Conclusion]This study enriches the research on endophyte within D.involucrate Brail,application of Bacillus bio-control,and therefore has laid a good foundation for the development of fungus used in biological control of crop pathogens.     

Effect of plant extracts on activity of some defense enzymes of apple fruit in interaction with Botrytis cinerea

The efficacy of seven plant extracts(neem,fennel,lavender,thyme,pennyroyal,salvia and asafetida) in controlling postharvest of apple(caused by Botrytis cinerea) was evaluated in vitro and in vivo.In vitro,all plant extracts treatments inhibited spore germination.Inhibitory rates of pore germination was 17.41 and 20.83% for neem extract treatment(methanolic and aqueous extracts,respectively) with significant difference compared to control(73.6 and 85.33%) for aqueous and methanol extracts.In the storage conditions,the application of aqueous extract of neem(at concentration of 25%) resulted in 89.11% reduction of disease severity compared with the untreated control.Results of enzymes activity showed the plant extracts can increase the activity of peroxidase,phenylalanine ammonia-lyase,β-1,3-glucanase and polyphenol oxidase in the presence of pathogens,in apple fruits.However,the results of this research revealed that application of neem extracts was more effective than the application of other plant extracts.According to this study,it could be concluded that plant extracts may be useful to control postharvest disease as a safe alternative option to chemical fungicides.     

Phylogenetic Analysis of Citrus tristeza virus Isolates of Wild Type Citrus in China

The genetic variation and phylogenetic relationships of Citrus tristeza virus （CTV） isolates collected from Chinese wild type citrus were analyzed by comparing the sequences of nine genomic regions （p23, p20, p13, p18, p25, p27, POL, HEL and k17） with the CTV isolates of cultivated citrus from different countries. The results showed that the divergence pattern of genomic RNA of the CTV isolates from wild type citrus was similar to that of other isolates from cultivated citrus, the 3′ proximal region was relatively conserved, and the 5′ proximal region had greater variability. The nine genomic regions of CTV isolates analyzed were found to have been under purifying selection in the evolution process. Phylogenetic analysis showed that the eleven Chinese wild CTV isolates were located at different clades and did not relfect their geographical origins, suggesting genetic diversity among the Chinese wild CTV populations. These results will aid in the understanding of molecular evolution of the Chinese CTV populations.     

Sources for Heat-Stable Resistance to Southern Root-Knot Nematode （Meloidogyne incognita） in Solanum lycopersicum

Southern root-knot nematode （Meloidogyne incognita） is a major problem in vegetable production in China due to the expansion of plastic tunnel and solar greenhouse. Using resistant cultivars is an effective approach to control the disease. Nine genes, Mi-1 to Mi-9, have been reported and only Mi-1 has been successfully used in tomato breeding. However, Mi-1 is inactive at a temperature above 28~C. In order to identify sources for heat-stable resistance to southern root-knot nematode, 53 genotypes of tomato （Solarium spp.） were inoculated with an isolate of M. incognita in the growth chamber at 28 or 32℃ for initial screening. 28 lines had less than 25 galls and were considered as resistant candidates. The top 60% （16 in total） of resistant candidates obtained from each temperature were subject to re-evaluation at 32~C using the same nematode isolate. Three lines ZN17, ZN 48, and LA0385 showed heat-stable resistance with an average of 10 galls or less per plant. LA0385 is a wild species, while ZNI7 and ZN48 are elite breeding lines. These lines were grown in a greenhouse for two seasons, and also showed high resistance with less than 10 galls per plant. Thus they were considered as good sources for breeding resistance to southern root-knot nematode in tomato.     

Characterization of Citrus tristeza virus Isolates by Indicators and Molecular Biology Methods

Citrus tristeza virus （CTV） exists in citrus as a large number of distinct strains differing in biological characters. The control strategies such as mild strains cross protection （MSCP） require a clear understanding of the characterization of CTV. For better understanding of the structure of CTV population and the relationship between molecular and biological characterization, 72 CTV samples collected from five provinces in China were studied, using biological indexing, p25/Hinf I restriction fragment length polymorphism （RFLP）, multiple molecular markers, and bidirectional RT-PCR assay. The mixture of severe stem pitting isolates was found to be dominant in the field. CTV isolates with p25/Hinf Ⅰ RFLP group 3 and p23/BD-PCR group Ⅰ, Ⅲ were the main cause of epidemics, and most CTV isolates were found to be the mixture of T30 and VT genotypes. More accurate identification of strain mixtures in the field and better understanding of the biological traits of the isolates may be achieved by applying the three molecular detection methods simultaneously.     

A Comparative Study on Mutagenic Effects of Space Flight and Irradiation of γ-rays on Rice

The experiment was conducted to study the mutagenic effects of space environment on seedling growth in M1 generation and plant height and heading date in M2 generation in rice. Two types of lines of growth promotion （GP） and growth suppression （GS） were selected from the recombinant inbred lines （RILs） derived from a cross between a sensitive japonica Lemont and a nonsensitive indica Teqing to compare the mutation frequency and mutagenic efficiency for plant height and heading date in the M2 generation. Space environment resulted in 34.9% higher seedling height （SH） in the GP group than in the control, and 39.1% lower in the GS group than in the control, and there was no difference in seed fertility （SF） between the two groups. In M2, mutants of plant height and heading date can be induced by space treatment in both the two groups with lower mutation frequency and mutagenic efficiency in the GP group than in the GS group. There were no significant differences in the physiological damages in M1 between the two groups after γ-rays irradiation treatment. Mutation frequency and mutagenic efficiency of heading date in M2 were higher in the GS group than in the GP group, and the opposite was true for mutagenic efficiency of plant height although the mutation frequency varied between the two groups. The mutation frequencies of plant height and heading date induced by space environment were obviously lower than those by γ-rays irradiation, but the mutagenic efficiency was the opposite for most of the traits. For the GP and GS of seedlings induced by space environment, the GS had higher mutation frequency and mutagenic efficiency for plant height and heading date than the GP in M2.     

Effects of Microtubule Polymerization Inhibitor on the Hypersensitive Response of Wheat Induced by the Non-Host Pathogen Sphaerotheca fuliginea

The plant cytoskeleton is a highly dynamic and versatile intracellular scaffold composed of microtubules and microfilaments, serving a multiplicity of functions in plant cells. To reveal the relationship between the cytoskeleton in wheat （Triticum aestivum L.） cv. Suwon 11 attacked by the non-host pathogen Sphaerotheca fuliginea and the initiation of the hypersensitive response, the microtubule inhibitor oryzalin was injected into the wheat leaves immediately prior to inoculation. The incidence of hypersensitive cell death was significantly lower than that in water-treated control. In addition, the occurrence of hypersensitive cell death was also delayed and S. fuliginea was able to penetrate and form haustoria in epidermal tissues of wheat. All the results above indicated that hypersensitive cell death was associated with depolymerisation of microtubules, suggesting that microtubules might play an important role in the expression of non-host resistance of wheat.     

Organic Compounds in Silica Bodies of Rice Revealed by X-ray Microanalysis and Micro-infrared Spectroscopy

To understand the possible mechanism controlling the formation of silicon bodies in higher plants, we need to know the nature of organic compounds associated with silicon bodies and induces silicon precipitation in plant cells. A new method was developed to isolate pure silicon body in fresh leaves of rice (Oryaa sativa L. Var. 297) at lowt emperature by grinding and centrifugation procedures, which avoided degradation of organic molecules induced by high temperature and strong oxidizing acids used in the traditional method. The energy dispersive X-ray spectrum under scanning electron microscope showed that there was a great amount of carbon in silicon bodies in addition to silicon and oxygen. Organic compounds intimately associated with silicon bodies were released by treatment with HF solution. Analysis of the organic compounds by micro-infrared spectroscopy revealed the presence of polyphenol and polysaccharide and a little protein.     

Expression of Chicken Toll-Like Receptors and Signal Adaptors in Spleen and Cecum of Young Chickens Infected with Eimeria tenella

Toll-like receptors （TLRs） are a group of highly conserved molecules which initiate the innate immune response to pathogens by recognizing structural motifs of microbes. Understanding the changes in chicken Toll-like receptors （ChTLRs） and signal adaptors expression that occur with Eimeria tenella infection will help to elucidate the molecular basis of immune control of coccidiosis caused by Eimeria. The present study detected the dynamic changes in the expression of ChTLRs and associated signal adaptors in the spleen and cecum ofE. tenella-infected chickens during the early stage of infection. The results showed that the expression peak for ChTLRs, MyD88 and TRIF occurred at 12 h post-infection （hpi）, ChTLR3, ChTLRI 5 and MyD88 mRNA expression in the spleen ofE. tenella infected chickens were significantly higher （P〈0.05） than that of negative control chickens, and there were similar tendencies of these molecules expression in the cecum and spleen of E. tenella-infected chickens. The expression of MyD88 was upregnlated at four time points in the cecum of E. tenella-infected chickens. The results of this study indicate that ChTLR3, ChTLR15 and MyD88 play a role in young chickens infected with E. tenella.