Identification of viral pathogens of Rehmannia glutinosa disease in Henan Province ZHANG Zhen chen, ZHANG Lifang, QIAO Qi, WANG Yong jiang, JIN Xiu lan
(Institute of Plant Protection£¬ Henan Academy of Agricultural Sciences, Zhengzhou 450002, China)

Abstract: Virus species infected Rehmannia glutinosa were detected by using ELISA, RT-PCR and nucleotide (nt) sequence analysis of coat protein (CP) gene. The results revealed that Tobacco mosaic virus (TMV) was the major virus infecting Rehmannia glutinosa in Henan Province. CP gene of TMV from Rehmannia glutinosa (TMVª²-RH) was cloned and sequenced. The sequence analysis showed that thent and amino acid (aa) sequence of TMVª²-RH CP were 86.5% and 94.3% identical to TMV-U1 strain, and were 76.3% to 88.5% and 79.3% to 95.0% identical to other TMV strains, respectively. According to the results of phylogenetic tree generated based on aa sequences of CP genes, it is supposed that TMV-RH may be a new strain of TMV.
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Key words£ºRehmannia glutinosa; virus disease; Tobacco mosaic virus; strain

Study on vegetative compatibility of Curvularia lunata isolated from corn in China GONG Guo-shu, YU Xia, YE Hua-zhi, TANG Zhi-yan, ZHANG Min, LIU Ping, CHEN Xi-qin (Agricultural College, Sichuan Agricultural University, Ya'an 625014, China)ª¤

Abstract: Forty -four isolates of Corn Curvularia leaf spot fungus (Curvularia lunata) were isolated from infected corn leaves collected from different regions in China. Vegetative compatibility of these isolates was assessed using different methods. By pairing isolates on potato dextrose agar (PDA) and observing contact reaction between expanding colonies, four reaction types were observed, but no clear barrage formations were found. Hyphal anastomosis patterns were examined by microscopy and electron microscopy. Of all pairing combinations, very few complete hyphal anastomosis was observed, but the anastomosisª²like types that one hyphae was infinitely close to another were commonly found under microscopy. However,under electron microscopy, a combination interaction could be observed where the two hyphal contacted. The results showed that no clear barrage formation and anastomosis signs occurred by isolate pairing. Nitrate nonª²utilizing(nit)mutants of C. lunata were used to study vegetativecompatibility of C. lunata.The isolates were cultured on potato sucrose agar(PSA) containing KClO3(1.5%-3.0%)to generate chlorate-resistant mutants(sectors). The result indicated that 2.0%-3.0% chlorate in KPS was effective for most isolates. Nit mutants could be selected from chlorateª²reistant sectors on Czapek but not on minimal medium(MM). A total of 240 stable nit mutants were selected from 3640 sectors on Czapek,in which 59.2% were classed as nit1, 39.2% as nit3, 0.8% as NitMand 0.8% as nitD (unable to utilize nitrate, nitrite and hypoxanthine). Among the 44 tested isolates, 24(55%)generated nit mutants, and only two isolates(isolate 124 and 155) generated NitM mutants. The genetically different mutants from each isolate were complementally paired in all possible combinations. The results indicated that NitM was complementary with both nit1 and nit3 derived from the same parental isolate, and the remaining mutant combinations were unable to complement. Both isolate 124 and 155 were selfcompatible. Employing the two NitM mutants as tester, 5 isolates were classed to VCG1 while other 5 to VCG2.The remains did not belong to any VCG because of the absence of their nit or NitM mutants. These results suggest that complementation tests between nitrate nonª²utilizing mutants may be more suitable to assign vegetative compatibility groups of C. lunata.ª¤

Key words£ºCurvularia lunata; Corn Curvularia leaf spot disease; vegetative compatibility; nitrate-nonutilizing(nit) mutants; isolate pairing; hyphal anastomosis
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Biological characteristics of Fusarium lateritium Nees CHAI Zhao-xiang£¬ LI Jin-hua£¬ XIN Jian-feng£¨Plant Protection Department£¬ Gansu Agricultural University£¬ Lanzhou 730070£¬China£©
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Abstract: F. lateritium Nees, pathogen of cucumber muskmelon fruit, could grow on 10 different kinds of media. Oatmeal agar was the best medium and water agar was the worst one for its growing among the solid media. Czapek's yeast extract fluid medium was the best for its mycelium growing .The fungus grew well between 10 and 30¡æ with optimum of 25¡æ, pH 4.98-9.18 with optimum of pH 7.38. The fungus growth was enhanced by fluorescent light but inhibited by ultraviolet light. The fungus could utilize monosaccharides, polysaccharides and other carbon sources and organic nitrogen such as NaNO3, (NH4)2SO4 as nitrogen sources. L-aminopropionic acid was the nitroen source supported greatest growth of the fungus. The fungus was killed
at 50¡æ for 10 min. The conidia could germinate at 10-30¡æ,RH 90%-100% and pH 4.98-9.18,with the optimal at 25¡æ, RH>90% , and pH 7.38.ª¤

Key words£º Fusarium lateritium Nees; biological
characteristics

Prokaryotic expression and immuno-localization of AC4 gene of Tobacco curly shoot virusª¤ LI Yun-qin, CUI Xiao-feng, HU Dong-wei, LI Gui-xin, ZHOU Xue-ping*
(Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China)

Abstract: The AC4 gene of Tobacco curly shoot virus (TbCSV) Y35 isolate was obtained from the infected tobacco plants by PCR and cloned into expression vector pET-30a to generate the recombinant plasmid pET-Y35AC4. The recombinant plasmid was introduced into Escherichia coli strain BL21(DE3) pLysS, and TbCSV AC4 fusion protein was expressed with the induction by IPTG. The AC4 fusion protein was purified with Ni2£«-NTA resin and its polyclonal antibody was produced in a rabbit. Immuno-gold labeling with the antibody against AC4 fusion protein indicated that TbCSV AC4 protein localized in the chloroplast and mitochondria of infected tobacco cells.ª¤
Key words£ºTobacco curly shoot virus; AC4 gene; prokaryotic expression; localization

Immunocytochemical localization of deoxynivalenol in infected wheat spikes by Fusarium graminearum
KANG Zhen-sheng1, HUANG Li-li1, H. BUCHENAUER2
(1College of Plant Protection£¬ Biotechnology Center, Northwest Sci-Tech University of Agriculture and Forestry, Yangling, Shaanxi 712100, China; 2Institute of Phytomedicine, University of Hohenheim, 70593, Stuttgart, Germany)ª¤

Abstract:Fusarium toxin deoxynivalenol (DON) was localized in wheat spikes during infection process of Fusarium graminearum by means of immunocytochemical labeling technique. Hyphae of the pathogen produced toxin when they grew on inner surfaces of lemma and palea as early as 24 h after inoculation, and the toxin was detected inside of host tissues at this time. In hyphal cells, the toxin was localized in cytoplasm, mitochondria and cell wall, while the toxin was detected in cell wall, chloroplasts, plasmalemma and endoplasmic reticulum in host cells. At the earlier infection stage (2 days after inoculation), the hyphae extended only intercellularly in the host tissues. With increase in concentration of the toxin in host tissues, a series of pathogenic changes in host cells occurred and the hyphae then colonized degenerated host cells intracellularly (3-4 days afterinoculation). These results suggested that DON might play an important role in infection, pathogenesis and colonization for Fusarium graminearum on wheat spikes. In addition, labeling for DON demonstrated that toxin can be translocated upwards and downwards through vascular bundle of rachis from the infected floret, much toxin was detected in the upper florets than in the lower florets.

Key words£ºTriticum aestivum; Fusarium graminearum; deoxynivalenol; immunocytochemistry

The pathogenic reaction of banana pesudostem cells to different races of vascular wilt fungus and their crude toxins XU Wen-yao, WU Xu-hui, YANG Jing-hui, YANG Kun (College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, China)
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Abstract: Banana vascular wilt is a severe disease caused by Fusarium oxysporum f. sp.cubense in south China. This study describes the pathogenic reaction of the banana cells upon the inoculation of spore suspension and crude toxins of F. oxysporum f. sp. cubense. The detached pesudostems and pesudostem cells of banana plantlets were treated with pathogen spores or crude toxin solutions, and the pathogenic reactions were observed by using tissue sectioning. The results
showed that the different races of the F. oxysporum f. sp. cubense had their specific host range when the plantlets were inoculated with the fungal spores. However, all the banana cultivars showed the same reactions, such as browning reaction, upon the inoculation of crude toxins produced by different fungal races. This result suggested that the virulence differentiation of F. oxysporum f. sp. cubense was determinated by some unknown factors rather than the toxin specificity. It is also proved that the toxins produced by F. oxysporum f. sp. cubense are nonª²selective for banana.
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Key words£ºBanana vascular wilt; Fusarium oxysporum f. sp. cubense; race; toxin; pathogenic reaction

RFLP analysis of ITS region of rDNA in the falcate-spored species of Colletotrichum
ZENG Da-xing1,2£¬ QI Pei-kun2£¬ JIANG Zi-de2(1 Department of Applied Biological Engineering, Shenzhen Polytechnic, Shenzhen 518055, China; 2 College of Natural Resources & Environment, South China Agricultural University, Guangzhou 510642, China£©
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Abstract: Genetic divergence among 38 strains belonging to the falcateª²spored species of Colletotrichum was assessed by RFLP analysis on the basis of rDNA ITS region. The PCR amplified ITS region (ITS4-ITS5) was about 650 bp in length in all the tested strains. RFLP patterns of ITS products digested with different endonucleases (Alu I, Bsu R I,Hin 6 I, Hpa II and Taq I) were not distinguishable within same species, but clearly different at the interspecies
level. UPGMA analysis of co-migrate band in restriction patterns showed that 38 isolates could be divided into 6 distinct groups. Some strains previously in the different species, such as C. truncatum, C. circinans and C. capsici,were closely grouped together on a cluster dengrogram, indicating that they were possible belong to the same species.
Key words£ºColletotrichum spp.; rDNA; RFLP; genetic relationships

Possible function of superoxide anion radical in rice blast disease control by tricyclazole
ZHANG Chuan-qing, ZHOU Ming-guo (Department of Pesticide Science, Nanjing Agricultural University, Nanjing 210095, China)

Abstract: Effect of tricyclazole on production of superoxide anion radical (O 2-) in rice and the possible function of this effect on blast disease control were studied. Results showed that tricyclazole could significantly induce O 2- production in rice during chemicalª²plantª²pathogen interaction. O 2- scavengers, mannitol and ascorbic acid, can directly inhibit the mycelium growth rate of Magnaporthe grisea in vitro and have an antagonic effect on tricyclazole when treated after inoculation. Exogenous H2O2 and O 2- have an inhibitory effect on spore germination of M. grisea, and tricycla zole notably promoted this effect£¬ and so did the diffusates collected from inoculated leaves pre-treated with tricyclazole. These suggested that promoting O 2- production in host and decreasing anti-oxidative capacity of pathogen also are possibl mechanisms of tricyclazole against blast disease.ª¤
Key words£º tricyclazole; rice; Magnaporthe grisea;
superoxide anion radical; action mechanism

Preliminary analysis of genetic diversity and population structure of Ustilaginoidea virensª¤
ZHOU Yong-li1, FAN Jin-juan1,3, ZENG Chao-zhen4,
LIU Xiao-zhou2, WANG Shu2, ZHAO Kai-jun1ª¤(1 Key Laboratory of Crop Genetic and Breeding of Ministry of Agriculture,Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 2 Institute of Plant Protection, Liaoning Academy of Agricultural Science, Shenyang 110161, China; 3 Department of Biology, Shenyang Agricultural University, Shenyang 110161, China; 4 Institute of Agriculture, Guangxi University, Nanning 530005, China)
Abstract: The genetic diversity and population structure of 56 strains of U. virens collected from different rice growing areas in 1992, 1996 and 2001 were assessed with RAPD (random amplified polymorphic DNA). A total of 223 bands were amplified with 32 primers selected from 160 random primers. The similarity coefficient was over 0.80 among most of the strains. Strains from Northern, Southern and central rice growing areas were not tightly clustered based on location and sampling time, and the preliminary analysis showed that the genetic diversity of U. virens in China was not significant. More strains from different fields in different years will be needed in further study to understand the population structure
and genetic relationship of this pathogen in China.
Key words£º Rice false smut; Ustilaginoidea virens;
RAPD; population structureª¤ª¤

Development of a molecular detection assay for the physiological race MFR of Puccinia triticinaª¤
PU Zhi-gang1,2, LIU Tai-guo1, ZHANG Min2, CHEN Wan-quan1* (1Institute of Plant Protection, CAAS, Beijing 100094, China; 2College of Agronomy, Sichuan Agricultural University, Yaan 625014, China)

Abstract: A total of twenty-three physiological races of Puccinia triticina from wheat growing-areas of China and Mexico were investigated for the DNA polymorphism
by amplified fragment length polymorphism (AFLP). Sixty-four pairs of AFLP primers were used to generate polymorphic bands, and one specific DNA fragment was amplified by M05/E03 primer combination in race MFR of Puccinia triticina. The specific DNA polymorphic fragment, a 325 base pair band, was excised and cloned into T-easy vector. Based on the cloning and sequencing, the AFLP-marker was converted to a sequence characterized amplified region (SCAR) marker. Sixty isolates of Puccinia triticina were tested with the SCAR marker,indicating that MFR race could be differentiated from the rest races by the specific DNA marker exactly. The results provide a useful aid to e
stablish the system of molecular detection for the physiological races of wheat rust fungi.ª¤
Key words£ºPuccinia triticina£»molecular marker£»
virulence£»amplified fragment length polymorphism (AFLP)

Climate-based regional classification for oversummering of Puccinia striiformis in China with GIS
MA Zhan-hong1, SHI Shou-ding1, JIANG Yu-ying2,
ZHAO Zhong-hua2(1 Department of Plant Pathology, China Agricultural University, Beijing 100094, China; 2 National Agriculture Technique Extend Service Centre, Beijing 100026, China)
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Abstract: Temperature was a considerable factor to affect the oversummering process of the wheat stripe rust pathogen P.striiformis. The high-limit temperature for the pathogen oversummering combined with relevant wheatª²growth information was used to classify the wheatª²growing regions for the pathogen oversummering in China by using the Geographic Information System (GIS). The study demonstrated that
the regions suitable to the pathogen oversummering in China are huge. The pathogenª²oversummering regions in Gansu, Sichun, Yunnan and Shaaxi Provinces are
conjunct. The maximum temperature in July and August was between 20 and 23¡æ in most parts of eastern Gansu Province with exception in some counties, and the possi
bility of pathogen oversummering in these regions is low. The most wheatª²growing regions in Tibet and Qinghai Province are suitable to pathogen oversummering.The regions suitable to pathogen oversummering in Guizhou Province are most likely conjunct with those in Yunnan Province to serve as an integrated area for pathogen oversummering. However, regions in Yunnan Province are geographically complicated, and feature of pathogen oversummering is still under investigation.
Key words£º Wheat stripe rust; GIS; pathogen oversummering; regional division

Inoculation techniques used for inducing Rice false smut efficiently ZHANG Jun-cheng1,2, CHEN Zhi-yi1, ZHANG Bing-xin3, LIU Yong-feng1, LU Fan1(1 Plant Protection Institute, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; 2 Agricultural College, Guangxi University, Nanning 530005, China; 3 Department of Plant Protection, Zhejiang University, Hangzhou 310029, China)
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Abstract: Artificial inoculation techniques for false smut of rice (caused by Ustilaginoidea virens (Cooke) Takahashi) was studied under field condition. Inoculation with three inocula indicated that chlamydospores collected and kept at -20¡æ last year could not cause false smut; thin-wall conidia prepared from liquid culture could cause the disease, and higher concentration of conidia resulted in higher percentage of diseased panicles, the severest disease was caused by mixture of hyphaª²fragment and thin-wall conidia. Length of growing period of pathogen influenced inoculation effect. Among three inoculation stages of rice, the case inoculated 6-9 d before emergence of panicle resulted in optimum diseased effect. Among three inoculation periods on a day, the case inoculated at 4:00-6:00 pm. induced severest disease. Potato juice could increase percentageof diseased panicles when it was added to inoculum. The inoculation technique advanced in this paper could induced false smut efficiently, the optimum percentage of diseased panicles, disease index, number of spore balls in a panicle were 100%, 93.96, 110, respectively. The inoculation technique could distinguish differences of varietal resistance.
Key words£º rice; Ustilaginoidea virens; inoculation
technique

Study on constitution of physiological race of Magnaporthe grisea in rice mixture fieldª¤
YANG Jing, HE Xia-hong, WANG Yun-yue, LI Zuo-sen,
ZHU You-yong*ª¤(The Ministry of Education Key Laboratory for Agricultural Biodiversity for Pest Management, Yunnan Agricultural University, Kunming 650201, China)

Influences of arbuscular mycorrhizal fungi on growth and Fusarium-wilt disease of watermelon in fieldª¤
LI Min1, LIU Run-jin1ª³, LI Xiao-lin2ª¤(1Mycorrhiza Laboratory, Laiyang Agricultural College, Laiyang, Shandong 265200, China; 2College of Resources and Environmental Sciences, China Agricultural University, Beijing 100094, China)

Identification of Watermelon mosaic disease in Hefei
ZHANG Da-wei1,2£¬ LI Shi-fang2£¬ FAN Wei-hong1£¬
CHENG Zhuo-min2£¬ WANG Jie1*ª¤(1 Laboratory of Plant Pathology£¬ Anhui Agricultural University£¬ Hefei 230036£¬ China£» 2 Institute of Plant Protection of Chinese Academy of Agricultural Sciences£¬ The State Key Lab for Biology of Plant Disease and Insect Pest£¬ Beijing 100094£¬ China)ª¤

Identification of a Cucumber mosaic virus isolated from Salvia miltorrhiza Bge. and its coat protein gene sequence analysis WU Zhi-ming1, PENG Wei-xin1,
WEN Chun-xiu1, ZHU Shui-fang2, XIE Xiao-liang1
( 1 Institute of Economic Crop, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050051, China; 2 Institute of Animal and Plant Quarantine, AQSIO, Beijing 100029, China)