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Research progress on antimicrobial mechanism and genetic engineering of Bacillus for plant diseases biocontrol CHEN Zhong-yi¹， ZHANG Jie¹， HUANG Da-fang^1,2 （¹ State Key Lab for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, CAAS, Beijing 100094, China； ²Biotechnology Research Institute, CAAS, Beijing 100081， China）

Abstract: Some bacteria in the genus Bacillus，which are both resistant to adverse environment and usually against bacterial and fungal pathogens, are among the dominant microorganisms in the soil and plant microecologial systems, and a lot of natural isolates with great potency have been screened and applied widely in biocontrol of plant diseases. The biocontrol mechanisms of Bacillus include antagonism，competition and induced plant systemic resistance. Antagonism of Bacillus mainly owed to the production of antimicrobial proteins, e.g. bacteriocin, chitinase, glucanase, etc., and antibiotics as well as antifungal volatiles synthesized by secondary metabolism pathways. Improvement of the expression level of native antimicrobial genes and coexpression of foreign insecticidal or antimicrobial genes in one cell were of the most efficient approaches to genetically modify biocontrol Bacillus with stronger activities against plant diseases or against both plant diseases and insect pests. With the rapid progress of genomics and proteomics programs, research and development on antimicrobial molecular mechanism and genetic engineering of Bacillus will be greatly accelerated in the future.

Key words: Bacillus； biocontrol of plant diseases； mechanisms of antimicroorganisms； genetic engineering

The signaling pathways of disease resistance in Arabidopsis LIU Shi-wang， WU Xue-long， GUO Ze-jian* (Biotechnology Institute, Zhejiang University, Hangzhou 310029, China)

Abstract: Arabidopsis thaliana is a model plant to study the interplays between plants and pathogens. Plants exhibiting resistance or causing disease follow the hypothesis of “gene for gene”, in which only when the products from the avirulence gene of pathogen and the resistance gene of host recognize each other, can plants arise a series of defense responses and show resistance, otherwise plants develop diseases. Small molecules such as salicylic acid, jasmonic acid, ethylene play important roles in the defense responses, and the signal transduction pathways can be divided according to their effects in a particular system of the pathogen and plant interactions. Their roles in systemic acquired resistance and induced systemic resistance will be discussed in detail.

Key words: Arabidopsis; signal molecule; hypersensitive response; systemic acquired resistance; induced systemic resistance

The current state of classification and nomenclature of plant viruses. FAN Zai-feng， LI Huai-fang (Department of Plant Pathology, China Agricultural University, Beijing 100094，China)

Research Articles

Study on Cercospora sojina Hara and its action to soybean CAO Yue-ping¹, LI Hai-ying², LIU Xue-min³, CHEN Shao-jiang⁴, ZHANG Li-juan⁵, YANG Qing-kai³ (¹Shanghai Jiaotong University, Shanghai 201101, China;² Heilongjiang University, Harbin 150086, China; ³Northeast Agricultural University, Harbin 150030, China; ⁴China Agricultural University, Beijing 100094, China;⁵ Hebei Agricultural University, Baoding 071001, China)

Abstract: Soybean frogeye leaf spot produced by Cercospora sojina Hara is a worldwide disease, which had caused heavy losses in soybean production in

China

. In order to improve the disease control and resistance breeding, the biological characteristics, infection cycle, incubation period and forecast of this disease were summarized, at the same time, the differentiation of the physiological races， the inheritance of resistance to this disease and the application of toxins were also discussed in this paper.

Key words: Cercospora sojina Hara; infection cycle; incubation period; physiological race; inheritance of resistance

Identification techniques for physiological differentiation of Curvularia lunata in maize CHEN Jie， YAN Hong-hai， GAO Zeng-gui， XUE

Chun-sheng， ZHUANG Jing-hua （Key Lab. of Northern Crop Dis. Immun., Ministry of Agric., College of Pl. Protec., Shenyang Agric. Univ., Shenyang 110161， China）

Abstract: Eight inbred lines were selected from more than 20 dominant maize cultivars and 18 major inbred lines as differential hosts to identify the physiological differentiation of Curvularia lunata, they were Shen135,78599-1, Mo17, 477, C8605, E28, 7922 and Huangzao 4. The identification of physiological differentiation during seedling period in greenhouse was consistent with that in adult period, so identification in seedling period was selected as a most effective method since it was easily operative and easy to control environmental factors. Twenty isolates of Curvularia lunata were identified in 8 differential hosts as 6 pathogenic differential types in which type A with higher pathogenicity was distributed widely in heavily occurring areas, however, type D with weak pathogenicity was distributed in less occurring area, therefore type A could be considered as the dominant pathogenic group. Other major factors such as humidity, temperature and nutrition, to some extent, influencing identification of physiological differentiation were also investigated, and which produced some remarkable effect on identification of physiological differentiation of pathogen by the mentioned factors. The result mentioned above provided a sound basis for breeding and identification of resistant cultivars, reasonable distribution of resistant cultivars and monitoring of pathogen differentiation.

Key words: corn Curvularia leaf spots; Curvularia lunata; physiological differentiation; differential host

Mating Type, Fungicide Sensitivity and Physiological Race of Phytophthora infestans Collected from Yunnan Province K. Y. RYU¹,LUO Wen-fu^{2 *},YANG

Yan-li²,GUO Li-yun³,GUO Hua-chun⁴,WANG Yi⁵,CHEN Hai- ru² (¹National Alpine Agriculture Experimental Station, RDA, Korea; ² Department of Plant Pathology,Yunnan Agricultural University, Kunming 650201, China; ³Department of Plant Pathology,China Agricultural University, Beijing 100094, China; ⁴College of Agriculture and Biotechnology,Yunnan AgriculturalUniversity, Kunming 650201, China; ⁵ Office of International Potato Center in Beijing, Beijing 100081, China)

Abstract: The mating type, chemical response and physiological race of Phytophthora infestans collected from Yunnan Province were determined. In total, 134 isolates of P.infestans were used in this study, among which 124 isolates collected from potato planted in autumn of 2000 in Luliang，Jianshui，Nanjian areas and 10 isolates collected from potato planted in spring of 1999-2000 in Kunming and Qujing areas. Among 18 isolates collected from Luliang County，3 isolates belonged to A₂ mating type，while 15 isolates belonged to A₁ mating type. Only A₁mating type was detected in 106 isolates collected from potato in autumn of 2000 in Jianshui, Nanjian and in 10 isolates collected from 1999 to 2000 in Kunming and Qujing areas. The response of 83 isolates to metalaxyl showed that 71.1% were sensitive, 16.9% were intermediate resistant, and 12.0% were resistant. The percentage of metalaxyl sensitive isolates is higher in Luliang，Kunming and Qujing than in Nanjian and Jianshui. All of 83 isolates detected were sensitive to dimethomorph. Results also showed that the physiological races in Yunnan Province consisted of races 3.4, 0, 3 and 4, representing 48.0%, 32.5%, 1.56% and 3.9% of the population, respectively. However, only 0 and 3.4 races were detected in Nanjian, while races 0, 3, 3.4 and 4 races were detected in Kunming and races 0, 3 and 3.4 were detected in Luliang. This indicated that population of P. infestans in Kunming and Luliang areas is more diversified in race composition than that in Nanjian area.

Key words: mating type； metalaxyl； dimethomorph； race； Phytophthora infestans

Study on vegetative compatibility and anastomosis group in Botrytis cinerea Pers. GAO Jun-ming, LIU Hui-ping, WANG Jian-ming,YAN Xiu-qin, LI Xin-feng, HAN Ju-cai (Agronomy College, Shanxi Agricultural University,Taigu, 030801, China)

Abstract: Vegetative compatibility was determined in 92 isolates of Botrytis cinerea Pers. collected from Jinzhong, Yuncheng, Linfen, Changzhi and Datong in Shanxi Province by confront culture and observed under microscope. The results showed that 92 isolates could be classified into 7 different anastomosis groups (AG1, AG2, AG3, AG4, AG5, AG6, AG7) according to the vegetative compatibility and incompatibility. Four of the anastomosis types were distributed in Jinzhong (AG1, AG2, AG3, AG4), 3 types in Yuncheng (AG1, AG2, AG5), 4 types in Linfen (AG1, AG2, AG3, AG6), 4 types in Changzhi (AG1, AG2, AG5 ,AG7) and 3 types in Datong (AG2, AG3, AG6). AG2 was a prevailing group and was widely distributed in Shanxi Province.

Key words: Botrytis cinerea; vegetative compatibility; anastomosis; anastomosis groups

AFLP analysis of pathotype of Verticillium dahliae of cotton ZOU Ya-fei， JIAN Gui-liang， MA Cun，SHI Lei-yan， WANG Li-ping， LIU Hui-jun (Institute of Plant Protection, CAAS, Beijing 100094, China)

Abstract： In greenhouse the pathotype of 41 strains of V. dahliae were studied by inoculating the seedlings of 4 cotton differentiators, Ejing 1(S)， Zhongmiansuo 12(T) ，Wen-5(R) and Tangmian 2(R). Forty-one strains were clustered into 2 groups: defoliating and nondefoliating strains. System cluster analysis based on 169 polymorphic bands from results amplified with 8 effective primers was used to generate a dendrogram. Forty-one strains of V. dahliae tested were clustered into 2 groups: 10 nondefoliating strains and 1 intermediate strain; 30 defoliating strains. According to dendrogram，discovered certain relevant relationship in the strains of different geographical source. Amplified fragment length polymorphism (AFLP) was performed on 41 strains of V. dahliae of cotton in search of DNA probes specific for nondefoliating strains. Two pairs of primers (screened from 25 pairs) were used for AFLP, E64（GACTGCGTACCAATTCGAC）, M53（GATGAGTCCTGAGTAACCG）and E49（GACTGCGTACCAATTCCAG）, M65（GATGAGTCCTGAGTAAGAG），and yielded bands of 433 base pairs(bp) and 110 bp which were amplified only from nondefoliating strains and named EM₄₃₃ and EM₁₁₀, respectively.

Key words： cotton； Verticillium dahliae； AFLP analysis； specific bands； pathotype

Sequence analysis of the cylindrical inclusion protein gene of Luotian isolate of Wheat yellow mosaic virus GENG Bo， HAN Cheng-gui， LI Da-wei， YU Jia-lin， LIU Yi (State Key Laboratory for Agrobiotechnology and Department of Plant Pathology,China Agricultural University, Beijing 100094, China)

Abstract: A cDNA clone containing cylindrical inclusion (CI) protein gene from Luotian isolate of Wheat yellow mosaic virus (WYMV) in Hubei Province was obtained by RT-PCR, and the nucleotide sequence of the CI gene was determined. The CI gene of this isolate comprised 1977 nucleotides and encoded 659 amino acids containing consensus nucleotide binding motif. When the CI gene sequence of the isolate was compared with those of previously reported WYMV isolates from Huangchuan in Henan, Ya'an in Sichuan, Yangzhou in Jiangsu and Japan, the nucleotide and amino acid sequence similarities ranged between 95.0% and 97.5%, and between 93.2% and 97.1%, respectively. The function of CI protein was also discussed in this paper.

Key words: Wheat yellow mosaic virus; cylindrical inclusion protein gene; cDNA cloning; sequence analysis

RT-PCR of 2b gene fragments of some CMV isolates from China and comparison with their sequences ZHUANG Mu， WANG Xiao-wu*， XIE Bing-yan*， ZHENG

Wen-guang， FENG Lan-xiang， FANG Zhi-yuan （Biotechnology Department， Institute of Vegetables and Flowers， CAAS， Beijing 100081， China）

Abstract: A simple and rapid method for detecting Cucumber mosaic virus (CMV) was developed. CMV 2b gene fragments (300 bp) of 9 different isolates were successfully amplified by one step RT-PCR from virus samples prepared by grinding in an extraction buffer. RT-PCR products of 9 isolates were cloned into pMD18-T vector and 7 of them were sequenced. The comparison of these sequences revealed that 2b gene fragments of different CMV isolates were highly conservative, and the identity of nucleotide and amino acid sequences were over 93% and 90%, respectively. It showed that all the Chinese isolates used belong to the subgroupⅠof CMV.

Key words: CMV； 2b gene； RT-PCR； PTGS

Development of a SCAR marker linked to avirulence gene AVR-Pik^m in rice blast fungus Magnaporthe grisea.LIU Jun-feng*, ZHANG Guo-zhen*, MA Qiu-juan, PENG You-liang# (The Key Laboratory of Molecular Plant Pathology, Ministry of Agriculture, and Department of Plant Pathology, China Agricultural University, Beijing 100094, China)

Abstract: A randomly amplified polymorphic DNA (RAPD) marker, OPO12₁₀₀₀, which is closely linked to the avirulence gene AVR-Pik^m conferring avirulence in Magnaporthe grisea on rice cultivar Tsuyuake, was cloned and sequenced. Sequence analysis showed that OPO12₁₀₀₀ was 946 bp in length and did not contain any sequence homologous with the previously reported Mg-SINE, Fosburry, Magyy, Grasshopper, Pot2 and Pot3. According to the sequence, a pair of 24 mer primers was designed and used in PCR with templates from an avirulent parental strain S1522, a virulent parental strain S159 and 108 progenies from the cross S1522×S159. The PCR amplification produced a single DNA band of about 1000 bp which was as the same as OPO12₁₀₀₀in the avirulent strains while no amplification was detected in the virulent strains except for 5 virulent recombinants. The results indicated that the RAPD marker, OPO12₁₀₀₀, was converted into a SCAR marker successfully and it could be used to initiate chromosome walking for cloning of the target gene.

Key words: Magnaporthe grisea; avirulence gene; RAPD marker; SCAR marker

Effects of cytoskeleton inhibitors on defense responses of rice to Magnaporthe grisea YANG Min-he^1，2， ZHENG Zhong²， Jan E. LEACH³ (¹ College of Agronomy, Jiangxi Agricultural University, Nanchang 330045, China; ² Department of Plant Protection, Zhejiang University, Hangzhou 310029, China; ³ Department of Plant Pathology, Kansas State University, Manhatton 66506, USA)

Abstract: Treatments of spore suspension of M. grisea with microtubule inhibitor oryzalin and two microfilament inhibitors cytochalasin A(CA) and cytochalasin D(CD) indicated that 5—50 μmol/L oryzalin, 0.5—1.0 μg/mL CA and 1—20 μg/mL CD treatment had no effects on spore germination and appresorium formation. When rice leaf sheath was treated with these inhibitors, an enhanced fungal development was observed in host cells compared with that in untreated rice leaf sheath after M. grisea inoculation. The enhancement of fungal development was associated with the delay and suppression of host defense responses such as cytoplasm aggregation, phenolic compounds accumulation and hypersensitive cell death. The results indicated that rice resistance to M. grisea invasion was closely related to microtubules and microfilaments in rice cells.

Key words: cytoskeleton; inhibitors; rice; defense responses

Study on the blast resistance of transgenic rice plants with two antifungal protein genes YANG Qi-yun¹, XU Xin-ping², ZHU Xiao-yuan¹, FENG

Dao-rong², LI Bao-jian² （¹ Plant Protection Research Institute,Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China; ²Biotechnology Research Center, Zhongshan University, Guangzhou 510275, China）

Abstract: Twenty two transgenic rice lines with basic chitinase gene and β-1，-glucanase gene were screened for blast resistance by conducting seedling screening, resistant spectrum test, and field resistance test in the blast zone nursery. A series of transgenic rice plants with improved resistance were obtained. Among them, seven transgenic lines with high resistance were selected from transgenic rice line

F4-9, and these highly resistant lines also expressed stable resistance in the R7 generations based on the tests of resistant spectrum to blast fungus in greenhouse and the neck resistance in blast nursery. The transgenic rice lines with high resistance and quality were obtained successfully in this experiment. The data showed that genetic engineering was a powerful approach to improve the resistance of rice variety to blast and alleviate the negative correlation between blast resistance and quality in southern rice area.

Key words: transgenic rice plants; basic chitinase gene; β-1，3-glucanase gene; resistance to rice blast

Effects of humidity regulation on the infection of Botrytis cinerea to tomato LI Bao-ju, CHEN Li-qin, MENG Wei-jun, WANG Fu-jian (Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081,

China

)

Abstract: High humidity was a primary determinant of the spore germination of Botrytis cinerea, the causal agent of tomato gray mould. At least 80% of relative humidity(RH) was needed for the spore germination, and the highest germination ratio could be obtained when the spores were placed in water drops. At optimal temperature, spores and hyphae could infect the tomato plant to develop softening and rot lesion when RH reached over 85% and 80% respectively. In one day, sustaining time of high humidity directly influenced the development of tomato gray mould, the pathogen could infect tomato successfully when sustained time of high humidity(RH＞85%) was longer than 8 hrs.

Key words: tomato gray mould; humidity

Biological control of capsicum anthracnose by endophytic Bacillus subtilis BS-2 and BS-1 HE Hong^1,2，CAI Xue-qing¹，GUAN Xiong¹，HU Fang-ping¹ (¹College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, China; ² Agricultural College of Zhanjiang Ocean University, Zhanjiang 524088, China)

Abstract: Two strains of the endophytic Bacillus subtilis BS-2 and BS-1 isolated from foliage and stem of capsicum respectively were used as biological control agents to control the anthracnose of capsicum. The results showed that the anthracnose of the capsicum seedlings and fruits caused by Colletotrichum gloeosporioides was controlled effectively by these two strains, with the control efficacy of

81.5%—93.3% and 66.1%—79.2％ for the seedling anthracnose, and 80.0%—100% and

60.0%—100% for the fruits anthracnose by BS-2 and BS-1 respectively. It also indicated that the growth of the capsicum seedlings was promoted significantly by the two strains. The effects of the disease control and growth promotion of BS-2 were better than those of the BS-1. The biological control mechanism of the two strains was discussed in this paper.

Key words: capsicum； anthracnose； endophytic Bacillus subtilis； biological control

Cell-lytic effect of Bacillus subtilis on plant fungal pathogens LIN Fu-cheng， LI De-bao (Institute of Microbiology, Life Sciences College, Zhejiang University, Hangzhou 310029, China)

Abstract: Nine hundred and seventy-six bacterial isolates were obtained in this study, among which a bacterial antagonistic isolate, Bacillus subtilis S9 from sugarcane rhizosphere showed cell-lytic effect on the plant pathogens, Rhizoctonia solani, Pythium ultimum and Fusarium oxysporum f.sp. niveum after 4-day-confronting incubation on PDA plates. However, S9 could not cause obvious inhibitory zones on PDA plates during this incubation. Lytic spots of hyphae of R. solani caused by S9 were clearly presented under scanning electronic microscope. The cell-lytic process of S9 is as follows: Bacterial cells of S9 absorb on hypae of R.solani firstly, and grow and reproduce as the fungal hyphae grow, and then secrete some cell-lytic substances to decompose the hyphae. However, this study revealed that S9 hardly interfered with the growth of the fungal antagonists, such as Trichoderma viride, Chaetomium cupreum and Chaetomium globosum. Pot experiments proved that S9 could effectively control root disease of tomato caused by R.solani. It suggested that S9 mixed with above fungal antagonists could potentially synergistically control plant diseases caused by pathogenic fungi.

Key words: Bacillus subtilis; cell-lytic effect; fungal antagonists; biocontrol

Preliminary study on the mechanism of resistance to metalaxyl in Phytophthora infestans LIU Hong-bin¹， GU Bao-gen²，　LIU Xi-li¹， ZHOU Li-gang¹，　LI Jian-qiang¹ (¹ Department of Plant Pathology, China Agricultural University, Beijing 100094, China;² Institute for Control of Agrochemicals, Ministry of Agriculture, Beijing 100026, China)

Abstract： Isolates of Phytophthora infestans, two sensitive, two intermediate, and two highly resistant ones to metalaxyl, were selected. The membrane permeability and endogenous RNA polymerase activities were examined by means of the conductivity and radioactive isotopic incorporation technique，metalaxyl decomposition was also tested by HPLC. The results showed that metalaxyl displayed no significant effects on the membrane permeability of the three kinds of isolates with different sensitivities to metalaxyl. RNA polymerase activities of the sensitive isolates were strongly inhibited as metalaxyl concentration increased. The inhibition rate was more than 40% when the concentration of metalaxyl was kept at 10 μg/mL. Metalaxyl slightly inhibited RNA polymerase activities of the highly resistant isolates, the inhibition percentage was lower than 8% and 21%, respectively, when the concentrations of metalaxyl were at 10μg/mL and 100μg/mL. The inhibition of RNA polymerase activities of intermediate isolates was between the sensitive isolates and highly resistant isolates. The resistant isolates HL105 and SC1 presented some effects on decomposition of metalaxyl, and the decomposition rate was 13.46% and 16.14%, 30 days after incubation of the isolates.

Key words: Phytophthora infestans； fungicide resistance； membrane permeability； RNA polymerase activity； decomposition of metalaxyl

Effects of different cultivation methods on the pepper postharvest diseases ZHANG Zhi-yuan^1，2，GUAN Chun-yun² （¹　Department of Applied Chemistry and Environmental Science, Changsha University, Changsha 410003, China；²Agricultural College, Hunan Agricultural University, Changsha 410128, China)

Abstract: The article contrasted effects of plastic film-covering cultivation and open land cultivation on the species of pathogens causing latent infection in pepper and their distribution in different positions of the pepper fruits, disease incidence of the pepper postharvest fruits, and species of pathogens. The percentage of latently infected fruits in plastic filmcovering cultivation is dropped by 6.7 times than open cultivation. Pathogens of latent infection distributed in fruit-stalk, fruit-base, fruit-pulp and the percentage of contaminated fruit-base and the disease is the highest in fruit-base; fruits surface sterilizing could decrease remarkably the incidence of disease of pepper postharvest before the pepper was stored; there are 5 kinds of pathogens in the pepper postharvest fruits: Fusarium sp., Alternaria alternata, Colletotrichum gloeosporioides, Erwinia carotovora subsp.　carotovora, Aspergillus flavus. Prevalent species are F.sp.,E.c.subsp.carotovora and Colletotrichum gloeosporioides. A.alternata, E.c.subsp. carotovora and Colletotrichum gloeosporioides could cause latent infections.

Key words: pepper; plastic film-covering cultivation； the postharvest disease

Notes

The cloning and sequences analysis of CP gene of the isolation of Carnation mottle virus in Yunnan Province KONG Bao-hua¹, LI Wen-jun², CHANG Sheng-jun², CAI Hong¹, LIU Jin-yuan², CHEN Hai-ru¹ （¹ Key Laboratory for Phytopathology of Yunnan Province, Kunming 650201, China; ²Department of Biology and Technology,

Tsinghua University, Beijing, 100084,

China

)

Colonization and population dynamics of Bacillus subtilis B501 in the rhizosphere of strawberry WANG Zhan-wu¹, LI Xiao-zhi¹, LIU Yan-li¹， TIAN Hong-tao² (¹Institute of Agrophysics Plant Physiology and Biochemistry, Hebei Academy of Agricultural and Forest Sciences, Shijiazhuang 050051,China; ²Institute of Food Science and Technology, Hebei Agricultural University, Baoding 071001,China)

Changes of the PAL activity and induced resistance to TMV in tobacco treated with SA and inoculated with TMV LIU Tai-guo^1,2, SHI Yan-xia³, WEN Jing-zhi¹, LI Yong-hao¹ (¹Department of Plant Protection, Northeast Agricultural University, Harbin 150030, China; ² Institute of Plant Protection, CAAS, Beijing 100094, China; 3 Institute of Vegetables and Flowers, CAAS, Beijing 100081, China)