10 February 2010, Volume 40 Issue 1
    

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  • RAPD analysis and SCAR marker establishment of Su11 group of Puccinia striiformis f. sp. tritici in China
    HAO Bao-jun, WANG Bao-tong, LI Qiang, LI Gao-bao, WANG Fang, ZHANG Bo, KANG Zhen-sheng
    Acta Phytopathologica Sinica. 2010, 40(1): 1-6.
    Abstract ( ) Download PDF ( )   Knowledge map   Save
    Suwonll (Su11) group has been becoming dominant in recent years. For simplification of identifying method, random amplified polymorphic DNA (RAPD) was used to analyze polymorphisms and find specific DNA band of eight pathotypes in the group. Of the 190 random primers analyzed in total, 94 primers firmly showed amplification profiles. The results indicated that the genetic variations among types of Su11 were abundance. The specific DNA fragment of Su11-4 was found by amplifying with primer S1410 and those of Su11-14 were found by amplifying with S1412 and S1304. The specific DNA fragment obtained with S1304 was cloned and sequenced. Based on the sequencing, a primer pair was designed and the SCAR marker for Su11-14 was obtained. The results indicated that a molecular identification system for Puccinia striiformis f. sp. tritici races could be established by means of searching specific RAPD fragments of different races.
  • Identification of the pathogen causing brown spot of Ziziphus jujuba Mill. cv. Hupingzao
    YU Zhan-jing, HOU Xiao-jie, CUI Jian-zhou, RAN Long-xian, LV Xiao-hong
    Acta Phytopathologica Sinica. 2010, 40(1): 7-13.
    Abstract ( ) Download PDF ( )   Knowledge map   Save
    One disease on jujube fruit was reported in Shanxi Province in recent years. The typical symptom was brown spots formed on the top or shoulder of fruits. The suspected pathogen was isolated from diseased fruits. After pathogenicity tests in lab and field and re-isolation of the pathogen, the strain CN535 was determined to be responsible for the disease. CN535 colony on PAD was 69.2-73.5 mm in diameter, with concentric rings in light-gray and dark-green color in seven days. The conidia, (22.5-40.0) μm× (8.0-13.5) μm, were obclavate and obpyriform, ellipsoidal in form and formed singly or in short chains, with a conical or cylindrical beak and transverse, longitudinal or oblique septa; they were showing the typical morphology of the genus Alternaria. Its rDNA ITS sequence had 100% similarity with those of A. alternata, A. tenuissima, A. longipes, A. mali and A. citri searched in GenBank. The species specific fragments of 341 bp and 450 bp were amplified with two pairs of sensitive primers AAF2/AAR3 and Aalt-F/Aalt-R. Based on the morphological characteristics and rDNA molecular analysis, the pathogen was finally identified as A. alternata (Fries) Keissler.
  • Laboratory evaluation methods of apple Valsa canker disease caused by Valsa ceratosperma sensu Kobayashi
    WEI Jie-ling, HUANG Li-li, GAO Zuo-peng, KE Xi-wang, KANG Zhen-sheng
    Acta Phytopathologica Sinica. 2010, 40(1): 14-20.
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    Valsa canker is one of the most destructive bark-rot diseases on trunk of apple tree in China. The aim of the research was to develop a simple but stable, efficient and easy method for disease evaluation in laboratory. Excised leaves, young shoots, fruits and twigs of ‘Fuji’ apple trees were inoculated with a virulent isolate 03-8 after making different wounds. The samples were kept at 25℃ in a saturated moisture box. No disease was found in uninoculated and/or unwounded samples, while lesions were occurred in treatments by wound inoculation. The type of wound affected the disease development greatly. Lesions developed much faster on upper side wound than lower side of the leave. No significant difference was observed between wounds pricking one time and ten times on upper side of the leave. Bigger lesions were formed on young shoots by pricking wound than leaf scar. There were significant differences (P<0.05) among wounds of pricking one time, ten times and removing partial peel on fruits. Also, the disease developed much faster on leaf, young shoot and fruit samples (1.5-2 days) than on the twigs (10 days). Furthermore, four different virulent isolates were used to test the reliability and stability of the method. The results showed that expanded leaves or young shoots were better materials than twigs and fruits because different lesions caused by the different isolates could be observed obviously in less than 3 days. Thus, the evaluation method for Valsa canker was suggested as follows:using detached expanded leave or young shoot as plant material in growing season, inoculating one pathogenic fungi disc on the upper side of leaf or surface of shoot after pricking one time with needle, keeping at 25℃ for 2 days in a satisfied moisture box, then measuring the lesion diameter. This me-thod could be used to assess materials in quantity at a short time, obtain good results from replication, screen resistant cultivar, test isolate virulence and chemical efficacy indoor.
  • Detection of four grapevine leafroll-associated viruses by multiplex RT-PCR
    PEI Guang-qian, DONG Ya-feng, ZHANG Zun-ping, FAN Xu-dong, LI Li-li
    Acta Phytopathologica Sinica. 2010, 40(1): 21-26.
    Abstract ( ) Download PDF ( )   Knowledge map   Save
    The grapevines infected with leafroll viruses were inferior in vigour and stress resistance, poor fruit coloring, delayed maturity and lower sugar content. Eleven grapevine leafroll-associated viruses (GLRaVs) had been isolated from the infected grapevines so far. To improve detecting efficiency and decrease the cost, a multiplex RT-PCR based on single RT-PCR detection of GLRaVs was applied to detecting four grapevine leafroll-associated viruses (GLRaV-1, 3, 4, 5). In this paper, the mainly factors affecting RT-PCR reaction was studied. The results showed that concentration of template, primer or Taq DNA polymerase, annealing temperature and reaction cycles all had considerable influence on the system, and the influence of extension time or dNTP concentration wasn't significant. The sequence identity of GLRaV-1 was 96% compared with AF195822, GLRaV-3 was 99% with AJ748524, GLRaV-4 was 99% with EU746619, and GLRaV-5 was 95% with EU815935 in GenBank. The multiplex RT-PCR was proved simple, rapid and sensitive for detection of these 4 GLRaVs.
  • Establishment and application of multiplex RT-PCR for simultaneous detection of five watermelon viruses ZYMV, WMV, TMV, SqMV and CMV
    WANG Wei-lin, ZHANG Hao, YU Xiang-quan, WU Yun-feng, ZHANG Wen-bo, ZHANG Chun-ping
    Acta Phytopathologica Sinica. 2010, 40(1): 27-32.
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    A multiplex RT-PCR (mRT-PCR) system was established for simultaneous detection on Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Tobacco mosaic virus (TMV), Squash mosaic virus (SqMV)and Cucumber mosaic virus (CMV)infected watermelon by using five sets of specific primers designed on the basis of conserved sequences of the 5 viruses. The concentrations of the primers, Mg2+, Taq DNA polymerase and dNTPs were examined and the PCR conditions including annealing temperature and amplification cycles were optimized. The results showed that expected fragments of 542 bp (ZYMV), 485 bp (WMV), 410 bp (TMV), 354 bp (SqMV) and 293 bp (CMV)were amplified and the mRT-PCR system was successfully established and the multiplex RT-PCR had been applied in practice, provided a simple, rapid and economic method for simultaneous detection of the five watermelon viruses.
  • Deletion analysis of fliExoo, the gene encoding a flagellar base body protein in Xanthomonas oryzae pv. oryzae
    FU Ben-zhong, WU Mao-sen, CHEN Hua-min, HE Chen-yang
    Acta Phytopathologica Sinica. 2010, 40(1): 33-39.
    Abstract ( ) Download PDF ( )   Knowledge map   Save
    To elucidate the function of fliExoo gene encoding a putative flagellar base body protein in Xanthomonas oryzae pv. oryzae (Xoo), the gene deletion mutant △fliExoo was generated by the GmR marker exchange from the wild-type strain PXO99A. Non-flagellum, cell precipitation in the culture and significantly attenuated motility on 0.3% semi-solid medium were observed in △fliExoo compared to those of PXO99A. While there were no apparent alterations in bacterial growth and activity of cellulase in vitro, significant decreases in extracellular polysaccharide (EPS) production and biofilm formation were found in △fliExoo. The pathogenicity on rice (Oryza sativa L cv. Nipponbare) and induction of hypersensitive response (HR) on tobacco (Nicotiana tabacum L.) were attenuated in △fliExoo. △fliExoo-C, the complementation strain restored the phenotypes of the mutant △fliExoo partially or completely. As a result, the fliExoo mutation not only influenced on flagellar motility, but also virulence-related phenotypes in Xoo.
  • Cloning and sequencing of genome of banana bunchy top virus Haikou isolate
    FENG Tuan-cheng, WANG Jian-hua, LIU Zhi-xin
    Acta Phytopathologica Sinica. 2010, 40(1): 40-50.
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    The genome of the isolate of Banana bunchy top virus (BBTV) from Haikou was cloned and sequenced by the polymerase chain reaction (PCR) with total DNA from banana pseudostem and leaf showing typical BBTV symptom as template. The isolate originated from banana (Musa sp.) was designated BBTV-Haikou. The sequence analysis indicated that six full-length segments were 1 106, 1 040, 1 058, 1 040, 1 013 and 1 082 nt, respectively. All the segments contained an intergenic region and an open reading frame (ORF) except the segment 1, which had a small ORF in the main ORF. In the intergenic region (IR), there were a stem-loop common region (CR-SL) and a major common region (CR-M), which shared 91.55% and 88.45% identity, respectively. Comparison of the viral sequence from different country showed that DNA1 sequence was more conservative than other components, but DNA2 sequence mutation rate was higher than others, sharing homologies of 93.08% and 75.67%, respectively. Nucleotide sequence analysis between DNA1 of the isolate and that of the Asian group, Pacific group of BBTV and the two isolates of ABTV (Abacá bunchy top virus)showed that DNA1 of BBTV-Haikou shared 93.1%-99.1%, 89.6%-90.7% and 76.2%-77.4% nucleotide sequence identity, also 93.4%~100% and 85.7% deduced amino acids identity in BBTV and ABTV, respectively. According to the Karan's method of classification, the BBTV-Haikou isolate was belonged to the Asian group.
  • Genetic analysis and molecular mapping of stripe rust resistance gene in European wheat cultivar Mega
    LI Yang, YUAN Xi-li, YAO Qiang, HE Miao-miao, JING Jin-xue
    Acta Phytopathologica Sinica. 2010, 40(1): 51-56.
    Abstract ( ) Download PDF ( )   Knowledge map   Save
    Mega, one of the European wheat cultivar, possessed effective resistance to the dominant races (CYR30, CYR31, CYR32, Su-4 and Su-14) of Puccinia striiformis f. sp. tritici in China at seeding stage. To identify and map new stripe rust resistance genes, seedlings of the parents, F1, F2, BC1 progeny derived from a cross between resistant cultivar Mega and susceptible cultivar Mingxian169 were tested with the race CYR30 of P. striiformis f. sp. tritici in greenhouse. Simple sequence repeat (SSR) techniques were used to develop molecular markers linked to the resistance gene in wheat cultivar Mega. 237 pairs of wheat SSR primers were used to test the two parents, as well as resistant and susceptible bulks. Subsequently, 2 SSR markers were employed for genotyping the F2 population. The results indicated that the stripe rust resistance in cultivar Mega was conferred by a single dominant gene, temporarily designated as YrMe, located closely to the chromosome 5BL and flanked by two SSR markers Barc232 and Wmc640, with the genetic distances of 3.7 cM and 8.6 cM, respectively. The research provided theoretical basis to wheat breeding in the use of Mega.
  • The comparative study of Potato virus Y HC-Pro, CI, NIb and CP gene-mediated virus resistance
    LIU Jing, CHEN Xue-mei, SONG Yun-zhi, WU Bin, ZHU Chang-xiang, WEN Fu-jiang
    Acta Phytopathologica Sinica. 2010, 40(1): 57-65.
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    Using specific primers designed according to the cloned genome sequence of Potato virus YN (PVYN), 400 bp cDNA fragments were amplified from 3' end of HC-Pro, CI, NIb and CP genes, respectively. The four cDNA fragments were inserted into binary vector pRCHS contained a intron derived from Chalcone synthase to construct the antisense vectors pRCHS-HC-Pro, pRCHS-CI, pRCHS-NIb and pRCHS-CP, containing intron splicing hpRNA (ihpRNA), respectively. Recombinant binary vectors were introduced into Tobacco NC89 mediated by Agrobacterium tumefaciens and transgenic plants were obtained. The virus resistance tests indicated that the proportion of PVY-resistant transgenic tobaccos with pRCHS-HC-Pro, pRCHS-CI, pRCHS-NIb and pRCHS-CP was 55.34%, 73.69%, 61.54% and 84.21%, respectively. Northern blot revealed an inverse correlation between transgenic transcript accumulation and virus resistance, and siRNA was detected in the presence of disease-resistant plants. It demonstrated that the resistance was RNA mediated.
  • Identification, disease-preventing role and growth-promoting effect of biocontrol strain P1 to potato bacteria ring rot
    WANG Rui-xia, HE Yun-chun, ZHAO Ting-chang, TIAN Hong-xian, LI Yin-fan, LIU Fei
    Acta Phytopathologica Sinica. 2010, 40(1): 66-73.
    Abstract ( ) Download PDF ( )   Knowledge map   Save
    The endophytic bacteria strain P1 was isolated from healthy potato tubers collected in potato bacterial ring rot field. Antagonistic tests showed that P1 strongly restricted growth of the pathogenic bacterium, Clavibacter michiganense subsp. sepedonicum. Strain P1 was preliminarily identified as Bacillus sp. based on the morphological, physiological and biochemical characteristics and then as B. megatherium by further contrast analysis of 16S rDNA sequence. The antibacterial substances extracted from P1 cultures were proven as crude protein, which were not sensitive to UV and showed the strongest activity at pH 7.0. The inhibitory activity of the crude protein decreased significantly when the temperature was higher than 80℃. Greenhouse tests indicated that strain P1 had a significant role in biocontrol against potato bacterial ring rot with the efficiency of 53.4% and in the promotion of seedling height, stem thickness, potato weight and the rate of commodity tubers.
  • Mechanism of biological control of Phytophthora blight in pepper by mangrove endophytic bacterium strain RS261
    LIU Feng, OU Xiong-chang, HE Hong, HU Han-qiao, GUAN Xiu-qiong
    Acta Phytopathologica Sinica. 2010, 40(1): 74-80.
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    The mechanism of biological control of Phytophthora blight by treatment with mangrove endophy-tic bacterium RS261 was studied. Tagged by the resistance to rifampicin (300 μg/mL), the strain RS261 could colonize many plants, such as capsicum, tomato, eggplant, et al. An obvious promotion on pepper growth occurred after watering roots with strain RS261. The metabolites of strain RS261 showed strong inhibitory activity against mycelial growth and sporangia production of P. capsici. Inoculation with P. capsici considerably increased the content of MDA and the activities of SOD, CAT and POD in the capsicum. However, when co-inoculated with strain RS261 and P. capsici at the same time, the MDA content and SOD, POD and CAT activities were all lower than those inoculated with pathogen only. In addition, treatment by co-inoculation increased PAL activity, and the induced PAL activity might enhance anti-disease capability of the plants.
  • Influence of continuous tomato-cropping on second-stage juveniles of root-knot nematode and free-living nematodes from rhizosphere soil in plastic greenhouse
    SHI Li-bo, WANG Zhen-hua, WU Hai-yan, Liu Jing
    Acta Phytopathologica Sinica. 2010, 40(1): 81-89.
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    Root-knot nematode (Meloidogyne sp.) is a destructive pest of vegetable and difficult to be ma-naged, especially in greenhouse of continuous planting tomato. For determining the influence of continuous planting on soil nematodes, the dynamics of second-stage juveniles (RKN J2) of root-knot nematode and free-living nematode in rhizosphere soil were investigated during the tomato growing season. The individuals of RKN J2 increased with the longer planting years. The number of J2 at 0~30 cm in 0 and 5-year soil was significantly less than that of other tested soil (P<0.05), the average means were 1.1 and 2.1 per 100 g dry soil. While in 8, 10 and 12-year soil, J2 were 154.9, 68.3 and 861.8 per 100 g dry soil, respectively. The population of J2 increased with soil depth, and mainly distributed in 20~30 cm soil layer. The number of free-living nematode also increased with depth of soil. There was no relationship between the time of con-tinuous cropping and the number of soil free living nematode. The percentage of RKN J2 in total soil nematode was lower in 0 and 5-year, Meloidogyne sp. was the rare group and kept relative stable population structure with the other soil nematodes. However, RKN J2 in 8, 10 and 12-year soil was the dominant group. The adverse trend was found between the number of free living nematode and RKN J2, the proportion of RKN J2 in total soil nematode increased with the time of continuous planting, and the ratio of free-living nematodes to RKN J2 decreased significantly (P<0.05).
  • Isolation and identification of a new pathogen causing soft rot of Phalaenopsis amabilis
    CHU Xiao-ling, YANG Bo
    Acta Phytopathologica Sinica. 2010, 40(1): 90-94.
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    Soft rot disease often affects Phalaenopsis amabilis during the growing season. However, the pathogen of the disease is remaining poorly studied. In this study, bacterial strain R1 was isolated from soft rot tissues in Wuhan. The pathogenic, morphological, physiological, biochemical tests and 16S rDNA sequence analysis were carried out. The homology of 16S rDNA sequence between strain R1 and Pseudomonas grimontii was 99.72%, and its physiological and biochemical properties were also similar to those of Pseudomonas grimontiis. All these evidences indicated that strain R1 could be identified as a novel strain of Pseudomonas grimontii. The pathogenicity of the novel isolate was proved according to the Koch's postulates. This is the first report that Pseudomonas grimontii can cause soft rot disease of Phalaenopsis amabilis.
  • Development of a multiplex RT-PCR detection method for three sweet potato potyviruses
    ZHANG Ye-hui, ZHANG Zhen-chen, JIANG Shi-jun, QIN Yan-hong, ZHANG De-sheng, QIAO Qi, WANG Yong-jiang
    Acta Phytopathologica Sinica. 2010, 40(1): 95-98.
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    A multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) method was developed for the simultaneous detection and discrimination of three sweet potato potyviruses:Sweet potato feathery mottle virus (SPFMV), Sweet potato latent virus (SPLV) and Sweet potato virus G (SPVG). Three compatible sets of primers specific for each virus were designed in conserved regions of the coat protein (CP) gene for use in multiplex RT-PCR assay, and producing three distinct fragments 300, 420, and 600 bp, indicating the presence of SPFMV, SPLV and SPVG respectively. The individual RT-PCR assays and the multiplex assay were optimized for highest sensitivity and specificity. This study fulfilled the need for rapid and specific sweet potato potyvirus diagnostic tool and that also had the potential for investigating the epidemiology of sweet viral diseases.
  • Changes of defensive enzymes and PR-1a gene expression of tobacco induced by chito-oligosaccharides
    SHANG Wen-jing, WU Yun-feng, ZHAO Xiao-ming, DU Yu-guang, SHANG Hong-sheng
    Acta Phytopathologica Sinica. 2010, 40(1): 99-102.
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    The activity change of defensive enzymes and PR-1a gene expression of tobacco (Nicotiana tabacum) seedling induced by chito-oligosaccharides were studied. The results showed that high level systemic acquired resistance (SAR) was expressed in tobacco plants treated with chito-oligosaccharides solution at the concentration of 50 μg/mL. PAL activity increased greatly with 2 peaks, the activity of SOD decreased initially followed by an increase with higher increment, and the activity of POD peaked early followed by a gentle fall in chito-oligosaccharide treated plants. The PR-1a gene was strongly expressed in tobacco due to systemic acquired resistance induced by chito-oligosaccharides. At 168 h after inoculation the expression quantity (co-pies/2 μL) of PR-1a gene was increased to 2 469.6 in treated tobacco leaf, reached 392.6% than that at 0 h after inoculation, it was increased 3.05 times of that in untreated control.
  • The sporulation of Clonostachys rosea strain 67-1 in submerged fermentation and its resistance to adversity
    ZHANG Bao-yuan, SUN Man-hong, ZHANG Yong-hua, XIE Xiang-ming, LI Shi-dong
    Acta Phytopathologica Sinica. 2010, 40(1): 103-105.
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    The growth and sporulation of Clonostachys rosea strain 67-1 in PD broth was observed and figured out. A large amount of submerged spores were obtained in shake flask with proprietary Czapek me-dium. Meanwhile, the colonies of strain 67-1were cultured in PDA plate and aerial spores were collected by elution. Resistances of submerged spores and aerial spores to high temperature, dry condition and UV treatment were determined. The results showed that the survival of the submerged spores kept in 60℃ for 30 min was 76.7%, while the aerial spores were hardly germinated in the same condition. After two weeks' drying treatment, the spore activities were 89.2% and 29.3%, respectively. Similarly, the activity of submerged spores was 72.6% and that of aerial spores was 19.7% when exposing to UV for 1 min. It is illuminated that deep submerged fermentation is more efficient than solid culture for strain 67-1 to produce chlamydospores, which act as main component in biopesticide mass production.
  • Sensitivity of population of Blumeria graminis f. sp. tritici isolates to temperature in 2008
    WAN Qiong, DING Ke-jian, DUAN Xia-yu, ZHOU Yi-lin
    Acta Phytopathologica Sinica. 2010, 40(1): 106-109.
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    The sensitivity of 113 isolates of wheat powdery mildew (Blumeria graminis f. sp. tritici) sampled from 6 provinces or cities in 2008 to temperature was tested by detached leaf segment method with setting up 5 different temperatures indoor. The results showed the mean ET50 (which represents the temperature that is required to obtain 50% of the maximum effect) of all isolates tested was 23.02℃. ET50 values of 17.70% isolates were more than 24℃. The highest and the lowest ET50 of isolates were 25.22℃ and 19.42℃, respectively. There were a certain differences for isolates sensitivity to temperature among different provinces or cites. It was also found that when temperature increased during 22-26℃, the latent period of isolates prolonged, and the latent period of different isolates was different at the same temperature, too. These results will provide a reference for the oversummering division of wheat powdery mildew, as well as the effect of climate to the disease.
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Superintendent：
China Association for Science and Technology
Sponsored by：
Chinese Society for Plant Pathology
China Agricultural University
Editor in Chief: FAN Jun
Started in 1955
ISSN 0412-0914
CN 11-2184/Q
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