Lesion mimic mutants are characterized as the formation of necrotic lesions in the absence of pathogens, and such genetic defects often result in enhanced resistance to pathogen infection and constitutive expression of defense response genes. About 200 rice lesion mimics were found by different strategies, 52 of which had been identified by the end of May, 2009. Six genes had been cloned from these identified mutants and encoded distinct proteins including heat stress transcription factor, E3 ubiquitin ligase, acyltransferase, cytoplasmic protein kinase, zinc finger protein and ankyrin repeats protein. Although none of these proteins appeared to be directly involved in plant defense pathways, most of the 41 lesion mimics exhibited the resistance to <i>Xanthomonas campestris</i> pv. <i>oryzae </i>or<i> Magnaporthe grisea</i> and constitutive expression of typical defense-response marker genes such as callose, autofluorescence martials, phytoalexin, reactive oxygen specie, and pathogen-related genes. It was indicated that these lesion mimic mutations activated the defense system in plants, but they probably functioned in distinct signaling pathways.  Thus, extensive studies of rice lesion mi-mics will promote the better understanding  of molecular mechanisms on the crop resistance to pathogens and genetically improve resistance breeding in crops. 

Real-time PCR was used for detection of Leptosphaeria maculans, the causative agent of oilseed phoma stem canker. Specific primers Lmb3/R2 and a TaqMan-MGB probe (Probe-M) specific to L. maculans were designed from the differentiation of ITS sequences between L. maculans and related species in the GenBank database. The results showed that all 22 isolates of L. maculans from oilseed samples from Austra-lia, Ukraine and Canada could get positive amplification with the limit of 4 pg DNA, and negative results for 30 isolates of L. biglobosa and the other 6 isolates of related species from oilseed samples. Real-time PCR could be especially useful for mass screening of oilseed sample and diagnosis of suspected isolate.

A suspected new disease of sweetpotato was observed at a number of sweetpotato production areas in Guangdong Province since 2006. The disease symptoms often appear as yellow leaves and black water-soaked rot  extending from the bottom to the top of stems  resulting in collapse and death of the entire plant. Bacterial strains were isolated from the diseased seedling stems of sweetpotato. Inoculation of sweetpotato seedlings with the isolates caused the same symptoms as naturally infected plants. The isolate H12 was identified as <i>Erwinia chrysanthemi</i> based on pathogenicity, morphological characteristics, culture patterns, physiological and biochemical reactions, and 16S rDNA gene sequences. There was no resistance on eight sweetpotato varieties inoculated with the pathogen. This is the first report of bacterial stem and root rot of sweetpoato in China.

TaqMan real-time PCR was developed to detect Burkholderia caryophylli. A pair of primers and a probe specific for B. caryophylli were designed according to its intergenic spacer region of 16S-23S ribosomal RNA. Among seven tested pathogenic bacteria, only B.caryophylli was detected. The results showed that a limit of 0.4 pg/μL of B.caryophylli DNA was detected, more specific and sensitive than routine PCR. The method can also apply to the detection of B. caryophylli in inoculated samples and it is useful for rapid diagnosis and quarantine purposes.

Typical symptoms of phytoplasma infection were observed in <i>Sasa fortunei</i> (van Houtte)Fiori du-ring a disease survey in Yangling. Using 16S rRNA universal phytoplasma primer pairs R16mF2／R16mR1and R16F2n／R16R2, 1.2 kb specific fragment was obtained from bamboo with symptoms of little leaf and stunt. The fragment was sequenced and subjected to RFLP and phylogenetic analyses. The strain obtained from bamboo was belonged to “<i>Candidatus</i> Phytoplasma asteris” and shared identities of more than 98％ with other members in this group. Group specific primer pairs R16（Ⅰ）F1／R16（Ⅰ）R1 and R16（Ⅴ）F1／R16（Ⅴ）R1 confirmed that the strain was a member of group “<i>Candidatus</i> Phytoplasma asteris”. And it also revealed that no complex infection was existed. RFLP analysis showed that the phytoplasma was a member of 16SrⅠ-B subgroup. This is the first report of phytoplasma infection in <i>S. fortunei</i> (van Houtte)Fiori.

Rotylenchulus reniformis is one of the most important vegetable nematodes, and has widely host range and distribution. Based on the morphological and molecular characters, a Rotylenchulus population from Hangzhou, Zhejiang, China, in vegetable crops was identified as R. reniformis. Most morphometrics of Hang-zhou population of R. reniformis were identical with type specimens and some variations were detected. PCR Amplification and sequencing of ITS and D2D3 region of 28S RNA gene found that the two sequence lengths were 808 bp and 786 bp, respectively. Sequence alignment of ITS region of Hangzhou population and other inter-populations of R. reniformis, showed that Hangzhou population had 99.5% to 100% similarity with the three amphimitic populations, whereas 92.4% with that of one parthenogenic populations. The D2D3 region sequence of 28S RNA gene of Hangzhou population had the similarity of 99.4% to that of a R. reniformis populaiton in GenBank. The host range of Hangzhou population of R. reniformis included fruit and vegetable crops in 16 genera of 11 families. This is the first report of the R. reniformis in Zhejiang Province.

Pine wilt caused by pine wood nematode, Bursaphelenchus xylophilus, is a serious disease for pines. In this paper, changes of some physio-biochemical indexes in stem of 3-4 years old Japanese black pine, Pinus thunbergii inoculated with B. xylophilus were studied. The results showed that after nematode invasion,the soluble carbohydrate in pine seedlings decreased, and it was more significant at the middle and late stages of disease development. The soluble protein and ascorbic acid in pine seedlings decreased gradually, and the free amino acid in pine seedlings also decreased. It was suggested that the content of soluble protein in stems of pine could be used for early diagnosis of the disease. Roles of the four physio-biochemical indexes in pathogenic mechanism of the disease were discussed.

Using reverse transcription-PCR (RT-PCR), full-length cDNA clones of Cucumber mosaic virus M strain (CMV-M) were constructed. Through pseudo-recombination between CMV-M (RNA2 and RNA3) and CMV-Fny genomic RNAs, the pseudo-recombinants F1M2F3, F1F2M3 and F1M2M3 were successful obtained. The seedings of Nicotiana tabacum cv. White Burley inoculated with pseudo-recombinants F1M2F3, F1F2M3 and F1M2M3 showed symptoms of necrosis, chlorosis, vein clearing, labefaction, linearization of leaf apex, etc. Based on comparison of symtoms between pseudo-recombinants and wild type virus, the key virulent factors were speculated. The key factor which induced mosaic was CP, while RNA2 of CMV-M and CMV-Fny was involved with necrosis and linearization of leaf apex respectively. Real-time fluorescence quantitative PCR (FQ-FCR) analysis indicated that symptom difference in N. tabacum cv. White Burley induced by CMV-M, CMV-Fny, F1M2F3, F1F2M3 and F1M2M3 was not related to the accumulation level of CMV genomic RNAs.

According to published nucleotide sequence, the primers were designed. The region of 3′-terminal 1 800 nts encompassing the part of NIb gene, coat protein (CP) gene and 3′ untranslated region (UTR) of Sweet potato vein mosaic virus (SPVMV) isolated from Henan Province was cloned and sequenced by RT-PCR. The sequencing showed that the CP gene was consisted of 996 nt and encoded 332 amino acid residues (FJ687211). The deduced amino acid sequence of CP gene was 95.2%-98.5% identical to other isolates published before, and was 97.9% identical to SPVMV-GD (AY459611) isolate. The CP gene was cloned into expression vector pET-28a（+）for over-expression in prokaryotic cells. The SDS-PAGE showed that about 41 kDa specific fusion protein was produced after induction by IPTG. The expressed protein was purified from SDS-PAGE and the antiserum against the protein was raised in rabbit. The antiserum could be used for specific detection of SPVMV from field samples of sweet potato with ACP-ELISA. This antiserum was used for detecting the samples of Ipomoea batatas and I.setosa inoculated with the field specimens from 14 provinces. The results indicated that the SPVMV was common in China.

H122 is one of translocation lines of Triticum aestivum and Psathyrostachys huashanica through cross and back-cross. To identify and map the gene(s) conferring resistance to stripe rust, H122 was inoculated with four Chinese prevalent races, CYR29, CYR31, CYR32 and CYR33, and two pathotypes, Su11-4 and Su11-11. Based on resistance identification results, CYR32, CYR33 and Su11-4 were selected to test the F1, F2 and BC1 generations from the cross H122/Mingxian 169. The results showed that H122 was resistant to all the tested races, and one dominant gene conferring resistance to CYR32, one recessive gene conferring resistance to CYR33 and one dominant gene conferring resistance to Su11-4 which was temporarily designated YrH122. Also, linkage group was constructed for YrH122 using 185 F2 populations. Among 258 SSR primer pairs, three SSR primers, Xbarc229, Xwmc339 and Xwmc93, were linked to YrH122 with genetic distance from 4.3 to 11.0 cM. According to the locations of linked markers, the resistance gene was located on chromosome 1DL. The results of back tests of the three SSR markers indicated that YrH122 was derived from P.huashanica. Further, based on the source of the gene YrH122,molecular detection and chromosome location, YrH122 might be a novel stripe rust resistance gene.

A total of 178 Magnaporthe oryzae (M. oryzae) isolates, collected from different rice-cropping districts of Heilongjiang Province in 2006, were tested for pathogenicity against 12 Japanese and 7 Chinese differential varieties (DVs), 24 rice monogenic lines (MLs) with different blast resistance genes and 6 local leading cultivars. The results showed that 104 Japanese races (pathotypes) were identified and the predominant ones were 077.7, 017.1, 017.5 and 037.5. Resistance gene Pi9(t) expressed the broadest resistance spectrum (on average 97.75%) to all the blast isolates tested, and was of the highest utilization value in rice resistance breeding. Resistant genes Piz-5 and Pi12(t) also showed high utilization values due to their resis-tance spectra were 78.09% and 78.65%. The results showed that some resistance gene could upgrade the blast resistance of rice varieties, for example, Pi9(t) and Piz-5 upgrade the blast resistance of Kong Yu 131; Pi5(t) and Pita-2 upgrade the blast resistance of Ken Dao 10; Pi9(t) and Pita upgrade the blast resistance of Shang Yu 397; Piz-5 and Pi12(t) upgrade the blast resistance of Ken Dao 12. Simultaneously, the new resis-tance resources will be explored extensively and the new broad-spectrum resistance genes will be transfered into leading cultivars purposefully.

A cross between Verticillium wilt resistant tomato variety 05046 and susceptible variety 051355 was made for mapping Verticillium wilt resistance gene(s). Through inoculation test on its F1 and F2 progeny, it was proved that Verticillium wilt resistance gene was a single recessive gene.With 545 AFLP primer combinations and 101 SSR primer combinations, AFLP and SSR analysis was performed on two parents,their F2 resis-tant and susceptible bulks and F2 progeny. Three new AFLP markers and one new SSR marker linked to the Verticillium wilt resistance gene-Ve gene were identified in the study. Three AFLP markers E66M84-A,E78M84-D and E66M40-A were mapped at 10.3 cM,14.2 cM and 30.5 cM genetic distance from the Ve gene respectively. The marker SSR599 was 12.5 cM away from the Ve gene.

An one-tube duplex real-time PCR approach for detecting Bean pod mottle virus (BPMV) and Tobacco ringspot virus (TRSV) was developed in this study. The solution of the same concentration of plasmids carrying either the CP gene of BPMV or that of TRSV was used as posivitive controls and the soybean seeds mix-infected by BPMV and TRSV were as experimental samples for real-time PCR detection. The results showed that both viruses could be detected in the same tube without cross-reaction. Although the detection limits for these two viruses were equivalent and up to 35 pg/mL in the positive samples, there were some differences in practical detection due to the uneven concentration of these two viruses in the soybean seeds. This approach suggested a simple, sensitive, rapid and more specific detection of BPMV and TRSV for the entry-exit inspection and quarantine.

Cloning approach of resistance gene analogs (RGA) is simple and practical method in finding plant resistant gene. In order to clone RGA from the foxtail millet cultivar Shilixiang resistant to rust , PCR method was carried out with degenerate primers based on conserved regions of NBS domain and STK domain. 25 NBS-LRR RGAs and 11 STK RGAs were isolated. The cloned NBS-LRR RGA had the conservative motifs P-loop, Kinase 2a, Kinase 3a and hydrophobic domain and the cloned STK RGA had eight catalytic domain. Tryptophan as the last residue of Kinase-2a motif and phylogenetic analysis showed that the cloned NBS-LRR RGAs were non-TIR NBS-LRR family. The further study of such RGA was useful for breeding of rust resis-tant foxtail millet cultivars and cloning rust resistant gene.

Three to five months old Fuji apple branches were inoculated with conidia or mycelium of Botryosphaeria dothidea. Barks were sampled around lenticels or tumors from the inoculated and naturally diseased branches. The samples were cut with freezing microtomy and microanatomy conformations of tumors were examined with the microtome-sections. Observation results showed that newly formed tumors could be found just under lenticels from the branches 30 days after inoculation. The mycelia grew and expanded radially from the lenticels and the hyphae in tumors were thick and strong with circular or truncated end. All hyphae in deve-loped tumors were surround with several layers suberized host cells. The suberized cells stretched out, arranged in rings and enclosed the hyphae. A suberous layer, constructed of several layers of suberized host cells and expanded from epidermal cells, could be found just around the tumor in later stage of tumor development. The results suggested that B. dothidea infection stimulated the production of new host phloem cells and the cell suberization. The new produced cells together with the mycelia formed the tumors. The suberous layer separated the tumor tissue from health host cell and resulted in necrosis of tumor and surrounded tissues. The edge of the necrotic tumor was then lift, like saddle next year and finally peeled off. The observation suggested that tumor forming and peeling were the results of host defending reaction to the pathogen.

In this study, the bait plasmid (pGBK-p24) with the full length of p24 encoded by Grapevine leafroll-associated virus 2 Liaoning isolate (GLRaV-2-LN）, and grapevine cDNA library were constructed, and then grapevine cDNA library was screened by the bait plasmid to identify protein(s) that interact with p24. The results showed that the aa sequence and open reading frame of the insert cDNA fragment of p24 were correct, and the bait plasmid has no self-activating effect on yeast strain AH109 and Y187; The cDNA library had a titer of 2.2×106 cfu/ mL, and most insert fragments were more than 700 bp in size , which indicated that the cDNA library are qualified for the yeast two-hybrid screening. 19 candidate postive clones displayed specific interaction with p24 and 16 cDNA sequences of these clones were obtained. BLAST showed that these cDNAs encode 8 proteins, such as MYC transcription factor, aconitase and type I inositol polyphosphate 5-phosphatase, etc; other three cDNA sequences encoded the same unnamed protein.

Huanglongbing(HLB,yellow shoot disease) has been a destructive disease of citrus around the world for over a century,yet the etiology of the disease has not been definitively established.Based mainly on assays for 16S rRNA gene sequences,fastidious bacteria known as "Candidatus Liberibacter species" have been associated with HLB.We are concerned that some current literature frequently refers to "Ca.Liberibac-ter spp." as the causal or etiological agent of HLB.However,Koch’s postulates,either sensu stricto or modified,have not yet been completely fulfilled to establish that "Ca.Liberibacter spp." are the cause of HLB.Direct pathological interactions between the bacteria and citrus host have not been conclusively documented.We suggest there is a need for the literature to be precise on this point until the etiology of HLB is firmly established.

Rice blast caused by Magnaporthe grisea is an important rice disease.It causes serious damage to both yield and grain quality.Early detection and monitoring of the pathogen is crucial for disease control.After inoculating the pathogen on the susceptible cultivar Menggu and the widely planted cultivar Hexi 39,the symptom was observed and the total DNA of the diseased rice tissues was purified and detected by real time PCR.It was showed that the little necrotic spot occurred at 72 hours after inoculation on Mengu and Hexi 39.The typical symptom was observed at 168 hours after inoculation on Menggu rice,but at 190 hours after inoculation on Hexi 39.The amount of the pathogen in both rice cultivars increased and reached the highest level at 48 hours after inoculation and decreased after 72 hours.There was some difference between susceptible and widely planted cultivar in pathogen DNA amount.Pathogen DNA in both rice cultivars could be detected firstly at 12 hours after inoculation,with 7.2×103 copies in Menggu and 4.9×103 copies in Hexi 39.The results proved that the real time PCR technique could be used to detect M.grisea at early stage of infection.

Banana sheath rot disease is a new disease of banana occurring in Guangdong,Guangxi,and Hainan Provinces of China in recent years.This study proved that the pathogen causing the disease is a kind of bacterium by testing of Koch′s postulates.The pathogenic bacterium was identified as Pantoea agglomerans based on pathogenicity,morphology,physiological and biochemical characteristics,host range,and sequence analysis of 16S rDNA.This is the first report that P.agglomerans caused the sheath rot of banana in China.

The gold immunochromatography technique was developed for the rapid detection of Pseudomonas syringae pv.lachrymans.Colloidal gold was obtained by reducing the gold chloride with sodium citrate,and 25 nm particles of colloidal gold were selected and labeled to polyclonal antibody(CMb) of P.syringae pv.lachrymans.CMb labeled with nanocolloidal gold particles was coated on the gold conjugate pad using the immune double sandwich method.The secondary antibody(goat anti-rabbit IgG antibody,targeting at the CMb) was coated on the surface of nitrocellulose filter membrane(NC) as the control line(C line),while CMb was coated on the surface of NC as the test line(T line),and the test strip was assembled.The prepared strip had a sensitivity of 106 cfu/mL and high specific,which was no cross reaction with including Pseudomonas fluorescens biovar Ⅱ and the other twenty-six strains.The working time of the test strip was about 15 minutes,and the strip stability test indicated that the result was reliable at 37℃ within 15 days.The test strip was used to detect samples of cucumber disease leaves collected from field,the C line and T line were observed clearly,while buffer solution control was negative reaction.The test strip can be applied to detect early bacterial angular leaf spot disease in cucumber production and direct the disease control.

The diseased samples of banana infected with CMV(Cucumber mosaic virus) were collected and a nucleic acid sequence based amplification(NASBA) detection system was developed.The specific primers were designed according to the high conserved region of RNA 2 sequence of CMV subgroupⅠ.The 310 bp specific amplification product was obtained in positive sample determined with 5% agarose gel electrophoresis.The 11 different banana samples were analyzed with NASBA and RT-PCR to find out reliability of the me-thod,the former was demonstrated by dot blot with digoxigenin-labeled probe and had the similar results with RT-PCR.Further,the sensitivities were also identical and was equal to 100 pg.

Tobacco plants are frequently infected by Tobacco mosaic virus(TMV),Cucumber mosaic virus(CMV),Tobacco etch virus(TEV),Potato virus Y(PVY) and Tobacco vein banding mosaic virus(TVBMV),and sometimes complexly infected in China.Five pairs of specific primers were designed according to the partial coat protein gene sequences of the five viruses.By optimization of primer,template concentration and amplification parameters,the expected fragments of 237 bp(TMV),273 bp(CMV),347 bp(TEV),456 bp(PVY) and 547 bp(TVBMV) were successfully amplified by the multiplex RT-PCR system.The results verified that this system was useful for simultaneous detection of these five viruses from field samples.

Sensitive and accurate detection of Potato spindle tuber viroid(PSTVd) is significant for seed potato production.Recombinant DNAs containing full-length monomeric,dimeric and partial-length PSTVd cDNA were constructed,and the various sized PSTVd-cDNAs were labeled by PCR amplification.For comparing the detection sensitivity and specificity of different labeled methods and reaction substrates,the monomeric PSTVd cDNA was also labeled with α-32P-UTP by transcriptional technique.Colorimetric and chemilumine-scent reactions in detection were also compared in N+-nylon membrane and Kodark film,respectively.The results showed that PSTVd dimeric probe,labeled with DIG and using CDP-Star substrate for chemiluminescent detection,produced the best sensitivity.The detection sensitivity was approximate 0.05 pg PSTVd total RNA,which is 500-fold of the previous published data.This detection system of NASH is high sensitive,specific and feasible for diagnosis of a large number of samples.

Antigenic membrane proteins(AMPs) encoded by phytoplasma play important roles in recognition between pathogen and host.To study the function of AMP of Paulownia witches′broom(PaWB) phytoplasma and detect the pahogen,specific primers were designed and used for amplification of the amp gene.The sequencing results showed that the open reading frame(ORF) of amp gene consisted of 696 nt and encoded 231 amino acid residues,which were 100% identical to two other sequenced amp genes of PaWB phytoplasma.For antibody preparation,the partial sequence of amp from nt 91 to 604 was cloned into the expression vector pGEX-4T-3.After induction by IPTG,the total protein was separated by SDS-PAGE,which showed that the fusion protein was overexpressed in E.coli.The antiserum against the fusion protein was raised in rabbit and applied specifically for detection of PaWB phytoplasma in Western blot,dot blot,ELISA,indirect immunofluorescence and immunocapture PCR assays.

The burrowing nematode,Radopholus similis is a quarantine nematode pest in the world.Using the universal primer pairs,the ITS region were amplified and sequenced,the ITS length were 706 bp,after analysis and alignment with known ITS sequences of R.similis.One species specific primer pairs,named RsF1/RsR1,was designed to diagnosis of burrowing nematode,the specific fragment was 271 bp.Combined the primer pairs D2A/D3B,the duplex PCR diagnosis for R.similis were developed to distinguish populations of R.similis from other plant parasitic nematode species,only R.similis populations could yield specie speci-fic 271 bp fragment,the other plant parasitic nematodes could not yield species specific fragment.

The presented assay of Cucumber mosaic virus strain(Cah1-CMV) contained full-length cloning,sequence analysis and host reaction tests,which was isolated from Canna in Hangzhou,China.RNA1 of Cah1-CMV was consisted of 3 356 nucleotides(nts),encoding 1a protein of 993 amino acids;RNA2 consisted of 3 045 nts,encoding 2a protein of 843 amino acids and 2b protein of 111 amino acids;and its RNA3 consisted of 2 220 nts,encoding 3a protein of 279 amino acids and CP protein of 218 amino acids,respectively.The results of phylogenetic analysis recommended that Cah1-CMV belonged to CMV subgroup IB.When inoculated onto typical CMV host plants,Cah1 induced systemic necrosis in N.glutinosa,but typical mosaic in other testing plants in Solanaceous Family.A recombinant virus FCah12b-CMV was constructed by replacing 2b gene of wild type Fny-CMV to that of Cah1-CMV.When FCah12b was inoculated on N.glutinosa,the top leaves were appearing brown and necrosis,similar to those induced by parental Cah1-CMV at the early stage of infection,but Fny-CMV did not induce similar symptom.At later infection stage,unlike Cah1-CMV,FCah12b-CMV did not cause systemic death.The Northern blotting showed that there was no significant dif-ference of viral RNA accumulation in systemic leaves infected by Cah1-CMV,Fny-CMV and FCah12b-CMV.The above results indicated that the 2b gene of Cah1-CMV played an important role in inducing viral symptoms,but it is not the determinant of symptoms on the whole plant.The differences in pathogenicity were not directly associated with the virus accumulation levels.

Magnaporthe oryzae(M.oryzae),a hemibiotrophic pathogenic fungus,can infect rice(Oryza sativa) and cause rice blast.The mechanisms involved in resistance of rice to blast have been studied extensively.However,it is difficult to investigate interactions between rice and M.oryzae because of the relatively large genome sequence of rice and the diversity of M.oryzae.The strain of M.oryzae,Y34,was found to be able to infect Arabidopsis thaliana Col-0 plants,so M.Oryzae(Y34)—Arabidopsis thaliana pathosystem was established to investigate the role of Arabidopsis thaliana ENHANCED DISEASE RESISTANCE 1(AtEDR1),which exerts negative regulation on powder mildew resistance by salicylic acid(SA) pathway.The results indicated that edr1 mutant was more susceptible to M.oryzae than wild type Col-0 plants.Previous double mutant analysis revealed that all edr1-associated disease resistance phenotypes were suppressed by mutants that block SA perception(nim1) or reduced SA production(pad4 and sid2).These double mutants were inoculated with Y34 to examine if they would suppress the susceptibility of edr1 to M.oryzae,and the results showed that compared with edr1,all double mutants exhibited resistance to M.oryzae.These data suggest AtEDR1 may play a positive role in the regulation of resistance to M.oryzae in Arabidopsis,which is required for SA pathway.

Bacillus subtilis strain BAB-1 exhibited an effective inhibitory activity against Botrytis cinerea which caused tomato gray mold.To clarify the chemical nature of the antagonistic compounds,lipopeptides were isolated from the cultural supernatant of strain BAB-1 by the methods of hydrochloric acid precipitation and methanol extraction.The lipopeptide compound showed a similar retention time to the standard sample of surfactin in HPLC and TLC analyses.Furthermore,ESI/MS analysis of the purified surfactant revealed 3 + ion peaks at m/z 1 009.3,m/z 1 023.4 and m/z 1 037.4 respectively,which were in agreement with the 3 homologous compounds of standard surfactin.Specific PCR amplification of the sfp gene fragment confirmed the existence of the surfactin biosynthetic genes in strain BAB-1.In addition,the purified surfactin had hemolytic and oil expansion activities,and its crystals appeared a cruciate structure under light microscope.

A biocontrol bacterium YC-10 was isolated from tobacco and tested for the nematicidal activity.The bacterium culture diluted 1,5,10,20 and 40 times all showed high nematicidal activity to Meloidogyne incognita and Heterodera glycines Ichinohe.After the nematodes were treated with the culture for 96 hours,the lethal rate of the M.incognita attained 100% and the H.glycines Ichinohe over 90%.The results indicated the potential application of biocontrol to nematode.The bacterium YC-10 was identified.

Identification of tuber rot pathogen on Stachys sieboldii Miq.was carried out with morphological and molecular methods for the first time.Though the observation of morphology and color of the colonies,morphology of macroconidia,microconidia and chlamydospores,it was preliminarily identified as Fusarium oxysporum.The fungus DNA was amplified by PCR with the universal primers of fungal rDNA ITS1 and ITS4,and sequenced.The sequencing results were logged in GenBank to BLAST,and all of the molecular

F2 populations were obtained from the cross between 9801-1 and DIANE.Bulked segregate and RAPD analyses were employed to identify molecules linked to the resistance to powdery mildew.OPP02 amplified about 792 bp polymorphic band in all individuals from 9801-1 and resistant bulk,but absent in all individuals from DIANE and susceptible bulk.By further analysis in F2 segregating population,the polymorphic band was found to be cosegregated with the resistant gene possibly.The fragment was sequenced,

Bacillus subtilis strain SB1 isolated from tomato roots was evaluated for its ability to control tomato bacterial wilt under greenhouse conditions.Application of strain SB1 culture suspension suppressed tomato wilt disease both in sterilized and non-sterilized soil,providing 76.03% and 77.78% protection,respectively.Wilt symptom was significantly reduced when strain SB1 was applied before pathogen inoculation,while less suppression was conferred by strain SB1 application after pathogen inoculati...

The pathogens of wheat black point were isolated and identified from 50 wheat varieties (lines) planted in Henan Province. The results showed that there were 4 fungal species, Alternaria alternate, Bipolaris sorokiniana, A. tenuissima and A. triticina, with 63.0%, 18.0%, 12.2% and 6.8% average isolation frequency respectively. And the isolation frequencies of various pathogens were dissimilar in different varieties (lines). Pathogenicity of these isolates was tested in field 10 days after florescence by spore suspension. The results indicated that A. alternate and B. sorokiniana had high pathogenicity. According to the isolation frequency and pathogenicity of the pathogens, A. alternate was considered as the dominant pathogen of wheat black point in Henan Province.

The cereal cyst nematode，Heterodera avenae，is an important nematode pathogen of wheat in China. The cellulose binding protein genes are the important parasitism genes for invasion of plant parasitic nematodes. The cDNA sequence of Ha-cbp-1 (GenBank accession GQ178086 ) was cloned by RACE kit based on homologous cloning method. The results showed that the cDNA sequence of Ha-cbp-1 contained an open rea-ding frame, which encoding 131 amino acids with a predicted signal peptide sequence for secretion and a cellulose-binding domain. The DNA sequence of Ha-cbp-1 contained two introns with the length of 932 bp. The predicted HA-CBP-1 amino acid sequence had 60% identity and 75%-76% similarity with HS-CBP-1 and HG-CBP-1.

Copper-resistance related genes copA and copB were amplified by using the DNA as template extracted from Xanthomonas citri subsp. citri. The target products were cloned, and sequencing results shows that sizes of copA and copB with 1 782 bp and 1 095 bp. The prokaryotic expression vectors pET-copA and pET-copB for these two genes were constructed. Both copA and copB were effectively expressed as fusion proteins in transformed Escherichia coli strain BL21(DE3) under the induction of IPTG. The expressed proteins were gel-purified and used to raise antisera in rabbits. The titers of antisera against recombinant proteins of copA and copB were 1∶6 400 in indirect-ELISA. The expressed products were subjected to Western blot analysis with prepared antisera. The results showed that the raised antisera reacted strongly with the antigenic proteins. These results indicated that the raised antisera could recognize specifically the recombinant proteins of copA and copB.

Two dimensional electrophoresis (2-DE) and MOLDI-TOF-TOF MS were used to analyse the proteomic changes in leaves of the susceptible cultivar WuYu3 and resistant cultivar KT95-418 rices infected with Rice strip virus (RSV). The results showed that disease specific protein (SP) of RSV accumulated more in susceptible cultivar WuYu3 than that in resistant cultivar KT95-418. The other 25 proteins were identified successfully by MS, including RSV NS2, host proteins related to photosynthesis, dynamic balance of cellular redox state or ions and protein translation/translocation or modification. The possible functions of these host proteins in the susceptible and resistant cultivars were discussed.

Sugarcane yellow leaf disease induced by Sugarcane yellow leaf virus (SCYLV) is a global viral disease. The coat protein gene (cp) that deduced 196 amino acids from CHN-FJ1 isolate was cloned by RT-PCR with the primers YLSCPF1and YLSCPR591. Sequence analysis showed that identity of nucleotides and deduced amino acids shared above 95% among the isolates from different geographical origins. The primers and TaqMan probe located on the conservative sequence of cp were designed for real-time fluorescent RT-PCR. The studies demonstrated that the detection limit was 1 000 copies/μL (3.61 fg/μL) of positive cloning plasmid, was 100-fold higher than that of regular RT-PCR. Typical amplification curve and Ct value did not appear in detection of Sugarcane mosaic virus (SCMV), the pathogens of sugarcane ratoon stunning di-sease (Leifsonia xyli subsp. xyli) and sugarcane smut (Ustilago scitaminea Syd.). The detection of leaf samples from field were conducted by real-time fluorescent RT-PCR, regular RT-PCR as well as tissue blot immunoassay (TBIA) and positive results reached 100%, 61.5% and 69.2%, respectively. The real-time fluorescent RT-PCR was more sensitive and reliable method for detection of SCYLV as compared with regular RT-PCR and TBIA.

This study was carried out to determine the induction of jasmonic acid (JA) on powdery mildew resistance in wheat, the activation of the expression of plant disease resistant marker genes （PR-1, PR-2 and PR-5）as well as a new cloned gene Ta-JA2, and to investigate the relationship between the induced resistance and the gene expression patterns. Susceptible cultivars “Chinese Spring”, “Pumai 9” and “Zhoumai18” were used to study wheat resistance activated by methyl jasmonate (MeJA) and powdery mildew resistance was then assessed with detached leaf assay. Real time quantitative RT-PCR was used to determine the expression patterns of PR-1, PR-2, PR-5 and Ta-JA2. The results demonstrated that MeJA application enhanced the powdery mildew resistance of “Chinese Spring”, “Pumai 9” and “Zhoumai 18”. The induced resistance could be detected from 12 h to 96 h after MeJA treatment, and the top value was at 24 h. MeJA significantly activated the expression of PR-1, PR-2, PR-5 and Ta-JA2. The induced powdery mildew resistance was positively associated with the induced expression of PR-1, PR-2, PR-5 and Ta-JA2. The phytohormone MeJA is a signal molecule in wheat powdery mildew resistance reaction.