Cloning of a Genomic DNA Encoding Caffeoyl-coenzyme A 3-<Emphasis Type="Italic">O</Emphasis>-methyltransferase of Citrus

In recent studies, we found that Apll (a PthA homologue) bound to three citrus proteins. Amino acid sequence analysis revealed that one of the target proteins was homologous to that of S-adenosyl-l-methionine : trans-caffeoyl-coenzyme A 3-O-methyltransferase (CCoAMT), an enzyme which is specific for the substrate trans-caffeoyl-CoA and catalyzes the synthesis of trans-feruloyl-CoA. From the consensus nucleotide sequences of CCoAMT genes, primers were chosen for PCR amplification of this gene from citrus total DNA. Two selected DNA fragments of 1.0 kb and 2.0 kb were obtained. These fragments were used as the probe to screen a citrus library. One clone, pCCl00, contained a 1.0-kb SalI fragment that hybridized to the probes. The nucleotide sequence of this fragment was determined in both directions. In this fragment, there was an open reading frame of 232 amino acids interrupted by an intron of 106 nt, and the deduced amino acid sequence had 95.9% homology to tobacco CCoAMT. Southern blot analysis of total citrus DNA showed that four EcoRI fragments hybridized to the probes, suggesting the presence of more than one copy of CCoAMT in the citrus DNA. Received 4 November 1999/ Accepted in revised form 26 November 1999     

β-1,3-D-Glucan Transported from Golgi Apparatus of Japanese Pear Leaves is a Component of Extracellular Polysaccharides Accumulated after AK-toxin I Treatment

This fluorescence and immunoelectron microscopic study showed that β-1,3-D-glucan accumulated only in leaves of a susceptible cultivar of Japanese pear after treatment with a host-specific toxin, AK-toxin I, from Alternate, alternata Japanese pear pathotype. The positive fluorescent reaction of callose was detected only in aniline blue fluorochrome-stained sections from toxin-treated leaves of the susceptible cultivar: positive sites were observed on cell walls of leaf cells. The sites of callose deposition were probably consistent spatially with modified sites on the plasma membrane that were observed only in the toxin-treated leaves of the susceptible cultivar. The toxin-induced modifications, identified as damage to the plasma membrane, were characterized by invagination of the plasmalemma specifically at plasmodesmata and as the concomitant accumulation of extracellular polysaccharides at the invaginated sites. A positive reaction to anti-β-1,3-D-glucan antibody was detected at the polysaccharides, Golgi vesicles, and trans-Golgi network (TGN) of toxin-treated leaves of the susceptible cultivar, but not at Golgi vesicles and TGN of water-treated ones. The cis-, medial and trans-Golgi stacks of toxin-treated leaves of the susceptible cultivar were negative for the antibody. The results showed that the polysaccharides, Golgi vesicles and TGN contained abundant β-1,3-D-glucan and that the glucan was transported from the Golgi apparatus via Golgi vesicles to the modified sites in cells of toxin-treated leaves of the susceptible cultivar. Received 7 March 2002/ Accepted in revised form 10 June 2002     

Importance of AC-rich Element on Pea Phenylalanine Ammonia-Lyase Gene 1 Promoter for Expression Induced by Nonpathogenic Attack

Regulatory elements in the promoter of phenylalanine ammonia-lyase gene 1 of pea (PSPAL1) in response to nonpathogenic attack were identified by in vivo footprinting analysis. The footprints determined AC-rich sequences, Box-I and Box-II, that were conserved at similar positions in the phenylpropanoid gene promoters from several plants. To reveal the functions of the AC-rich sequence in nonpathogen-responsiveness, we constructed Box-I-deletion PSPAL1 promoter (dB-1) with GUS reporter gene and transformed it into tobacco plant. The dB-1 had reduced basal expression and a complete loss of nonpathogen-responsiveness. These results indicate the essentiality of Box-I for PSPAL1 activation induced by nonpathogenic attack. Received 27 October 1999/ Accepted in revised form 25 November 1999     

海洋放线菌A3202的分离鉴定及其对柑橘采后病害的防效

为获得用于防治柑橘采后病害的海洋放线菌,以柑橘青霉病菌Penicillium italicum、柑橘绿霉病菌P.digitatum和柑橘炭疽病菌Colletotrichum gloeosporioides为指示菌,对分离到的25株海洋放线菌进行筛选。通过平板对峙法测定拮抗菌株的抑菌谱,并测定其发酵产物粗提物对柑橘果实采后病害的防效,根据形态特征、培养特征、生理生化特征及16S rDNA序列对其进行鉴定。结果表明,分离自鳞笠藤壶Tetraclita squamosa的菌株A3202对3种指示菌均具有强抑菌活性,对供试13种植物病原菌的菌丝生长具有不同程度的抑制作用,尤其对柑橘青霉病菌和柑橘绿霉病菌的抑制作用最强,抑菌带宽度分别可达2.33±0.05 cm和2.32±0.10 cm。该菌株800 mg/L粗提物溶液对接种后7 d的柑橘青霉病、柑橘绿霉病和柑橘炭疽病的相对防效均可达100%。初步鉴定该菌株为放线菌中的米修链霉菌Streptomyces misionensis。     

Functional Characterization of Citrus Polygalacturonase-inhibiting Protein

A cDNA encoding a polygalacturonase-inhibiting protein gene (SaiPGIPA) was identified from the citrus cultivar Sainumphung (Citrus sp.), one of the most popular cultivars in northern Thailand. SaiPGIPA was expressed in Escherichia coli cells, and the functional properties of citrus PGIP were analyzed. The PGIP fusion protein inhibited by a maximum of about 60% of the endopolygalacturonase activity, and a mixture of the PGIP and fungal endopoly-galacturonase released oligogalacturonides from polygalacturonic acid. The mixture containing the oligogalactur-onides, endopolygalacturonase and PGIP induced expression of the PGIP gene and a chalcone synthase gene in citrus leaves. The mixture also induced resistance in cucumber leaves against Colletotrichum lagenarium. Received 5 September 2001/ Accepted in revised form 20 November 2001     

Rhizoctonia Blight of Yacon Caused by Rhizoctonia solani AG-1 (IB)

Severe blight of stems and leaves caused by Rhizoctonia solani AG-1 (IB) was found on yacon (Smallanthus sonchifolius), a Compositae tuber crop, grown in Ehime Prefecture, Japan, from August to September 1996 and 1999. We named it “Rhizoctonia blight of yacon” as a new disease. Received 15 October 2001/ Accepted in revised form 25 November 2001     

Partial Purification of Thai Isolate of Citrus Huanglongbing (Greening) Bacterium and Antiserum Production for Serological Diagnosis

We aimed to improve the purification of citrus Huanglongbing (greening) bacterium (HB), Candidatus Liberobacter asiaticum and to produce an antiserum against HB. Periwinkle plants Catharantus roseum L. graft-inoculated with HB were used to produce an antiserum. All young leaves of new shoots incubated at 20–25°C and 25–30°C, a few mature leaves incubated at 20–25°C, and all mature leaves incubated first at 25–30°C and later transferred to 20–25°C developed yellowing symptoms and were then used to prepare immunogen. The HB was partially purified from these leaves by an improved method that included a macerating enzyme treatment of the midribs of infected leaves and homogenization of infected phloem sieve tissues. An antiserum raised against partially purified HB reacted clearly at a dilution of 1/16 with HB-infected citrus extract prepared at a concentration of 40 times, but did not react with healthy or tristeza virus-infected citrus extract in microprecipitin tests. Received 23 August 2002/ Accepted in revised form 4 December 2002     

短短芽胞杆菌对植物病原真菌的抑菌活性和抑菌机理

为探讨短短芽胞杆菌菌株011对植物病原真菌的防控效果,采用平皿打孔法测定菌株011粗提液对14种植物病原真菌的抑制作用,并以番茄早疫病为研究对象,研究了其抑菌机理。结果显示,菌株011粗提液对14种病原真菌均有抑菌活性,其中对番茄早疫病菌的抑菌活性最强,抑菌率达45.9%;菌株011粗提液能导致番茄早疫病菌菌丝生长异常,抑制孢子萌发;对番茄早疫病菌菌丝生长的EC50为71.4 mg/m L,对孢子萌发的EC50和MIC分别为0.12 mg/m L和0.8 mg/m L;该粗提液能引起番茄早疫病菌菌丝的脂质过氧化程度和细胞内过氧化氢含量上升,菌丝细胞膜的蛋白质合成量下降,当粗提液浓度高于50 mg/m L时,菌丝细胞膜的麦角甾醇合成量下降;该粗提液还能降低番茄早疫病菌胞外果胶酶和纤维素酶的活性。研究表明,菌株011粗提液可作为生防制剂应用于植物病原真菌的防治。     

Biotin Induces Sporulation of Mulberry Anthracnose Fungus, Colletotrichum dematium

Compounds in mulberry leaves inducing sporulation of C. dematium, mulberry anthracnose fungus, were detected. Dissolved or suspended aqueous solutions (1%) of 16 amino acids and 10 vitamins occurring in mulberry leaves were applied individually at the margin of the fungal colony growing on PSA plate. Sporulation was induced only where a biotin solution was applied on the mycelium at a concentration of at least 0.01 ppm. This result suggests that biotin, which occurs in mulberry leaves (ca. 0.6 mg/kg of dried leaves), has a role in inducing sporulation of C. dematium. Received 20 August 1999/ Accepted in revised form 16 November 1999     

Geminate Particle Morphology of Sweet Potato Leaf Curl Virus in Partially Purified Preparation and Its Serological Relationship to Two Begomoviruses by Western Blotting

Sweet potato leaf curl virus (SPLCV) is a possible member of the genus Begomovirus; however, the presence of typical geminate particles in sap has not been confirmed. We attempted to observe SPLCV virions by partially purifying the virus using an enzyme-assisted procedure. The observed virions in the partially purified preparations were typical geminate particles with a size of ca. 18×30nm. This virus preparation was subjected to western blot analysis using antisera against Bean golden mosaic virus (BGMV) and Mungbean yellow mosaic virus (MYMV). SPLCV reacted with both antisera. Specific bands appeared to be slightly larger than the 30-kDa marker protein and were considered to be SPLCV coat protein. This western blot analysis revealed for the first time a serelogical relationship between SPLCV and the two well-characterized begomoviruses. Received 28 June 1999/ Accepted in revised form 17 November 1999     

Rate, placement and timing of aldicarb applications to control Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), in oranges

BACKGROUND: The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a cosmopolitan insect pest of citrus and vectors the bacterium Candidatus Liberibacter asiaticus, a suspected causal organism of citrus greening or ‘huanglongbing’ disease. Aldicarb 150 g kg^?1 GR (Temik^® 15 G) was evaluated at three rates, two placements and three timings for ACP control in orange trees. RESULTS: Application of aldicarb at 5.6, 2.8 and 1.4 kg AI ha^?1 in March 2006 reduced adults by 58–66%, 45–46% and 25–37% respectively compared with untreated controls in two separate trials. No difference was observed in placement (one versus two sides of the tree) or tree size (8 years old versus 12 years old). Application at 5.6 kg ha^?1 in January 2007 reduced adults by 86% and shoot infestation by 77% in spring, and was generally better than the November and especially February applications. Even more striking results were evident on adults caged on treated plants for 25 days in March. Spiders and ladybeetles were equally abundant in treated and untreated trees. CONCLUSION: Aldicarb application at 5.6 kg ha^?1 to the bed side of mature citrus trees 2–3 months before spring growth can suppress ACP through spring without a direct effect on principal psyllid natural enemies. Copyright © 2008 Society of Chemical Industry     

Genomic Structure of a Geminivirus in the Genus Begomovirus from Yellow Vein-affected Eupatorium makinor

A virus from yellow vein-affected Eupatorium makinoi was tentatively designated as Eupatorium-infecting geminivirus (EuGV) on the basis of whitefly transmissibility, electron microscopic observation of geminate particles in sap and symptomatology. EuGV-speciflc DNA fragments were obtained by polymerase chain reaction (PCR). Based on the restriction analysis of the PCR products, EuGV was suggested to have a monopartite genome. We determined the putative, complete nucleotide sequence of the EuGV genome which is comprised of 2766 nucleotides. The EuGV sequence had two virus sense open reading frames (ORF)(V1, V2), four complementary sense ORFs (C1–C4) and a non-coding region termed the intergenic region. This genome structure is quite similar to other monopartite begomoviruses already reported. The nucleotide and amino acid sequences were compared with other begomoviruses. The results supported the conclusion that EuGV is distinct and divergent from other begomoviruses, whereas potential sequence motifs reported in other geminiviruses are well conserved. Received 17 March 1999/ Accepted in revised form 12 November 1999     

Comparative Analysis of Japanese and Foreign Strains of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> Based on 16S Ribosomal RNA Gene Sequences

We determined nearly the complete sequences of the 16S ribosomal RNA gene (rDNA) for Japanese strains of R. solanacearum. The comparison of 1471 nucleotide positions separated the Japanese strains into two groups, group 1 with biovars 1, 2, 3 and 4 strains which belonged to race 1, and group 2 with biovar 2 strains corresponding to race 3. Group 1 strains all had identical sequences, and strains representing the four biovars within the group did not differ from each other. Group 2 strains had characteristic nucleotides which differed at seven positions from group 1 strains. Comparative analysis of Japanese and foreign strains based on 16S rDNA sequences showed that Japanese group 1 was closely related to Asian and Australian biovars 3, 4 and 5, and belonged to the known division 1. Japanese group 2 was homogeneous to Indonesian biovars 2 and N2 in subdivision 2b. Since the differences in the nucleotides corresponded to restriction sites for the AluI, RFLP analysis of PCR-amplified 16S rDNA efficiently differentiated not only Japanese group 1 from group 2, but also differentiated three types of foreign strains which differed in biovar and geographic origin. Received 26 July 1999/ Accepted in revised form 19 November 1999     

Red Stripe of Rice Is Caused by a Bacterium <Emphasis Type="Italic">Microbacterium</Emphasis> sp.

In an etiological study to identify the causal agent of red stripe of rice, white bacterial colonies were consistently isolated from all diseased leaf samples collected in Indonesia. All the bacterial isolates were pathogenic to rice and caused symptoms similar to red stripe. The bacterial infection in rice leaves was confirmed histologically. Bacterial masses were observed in the lumina of xylem vessels in the lesion area, especially in those of connecting strands. Analysis of the 16S rDNA of the causal bacterium gave best matches to the 16S rDNA sequence of Microbacterium spp. of Microbacteriaceae which includes Clavibacter. Received 9 September 1999/ Accepted in revised form 16 November 1999     

Foot Rot of Ulluco Caused by Pythium aphanidermatum

Severe rot of stem bases caused by Pythium aphanidermatum was found on ulluco (Ullucus tuberosus) grown in Kagawa Prefecture, Japan, in September 1999. The name “foot rot of ulluco” is proposed for this new disease. Received 14 November 2001/ Accepted in revised form 7 January 2002     

<Emphasis Type="Italic">Pseudomonas syringae </Emphasis>Strains Are Classified into Five Groups by Comparing DMA Homology at the <Emphasis Type="Italic">hrp </Emphasis>Neighboring Regions

Previously, we classified Pseudomonas syringae strains into at least three groups (I, II and U) by comparing DNA homology at the hrp cluster and its neighboring regions (Inoue and Takikawa 1999). However, heterogeneous strains remained in the undetermined group (group U). We further classify group U, using pvs. syringae and coronafaciens as references. Comparison of restriction sites for regions of each pathovar revealed distinct differences. By using probes from the two pathovars, comparisons of DNA homology at the regions separated two additional distinct groups (III and IV) from group U. Therefore, P. syringae strains are classified into at least five groups. Received 4 November 1999/ Accepted in revised form 27 January 2000     

Shoot Blight and Leaf Spot of Blueberry Anthracnose Caused by <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis>

Shoot blight and leaf spots were found on highbush blueberry trees in Tsukuba, Ibaraki, in 1999. The causal fungus was identified morphologically as Colletotrichum acutatum Simmonds ex Simmonds. This is the first report of blueberry anthracnose caused by C. acutatum in Japan. Received 16 November 2001/ Accepted in revised form 22 March 2002     

21种药用植物提取物对柑橘青霉病抑菌作用的筛选及白薇醇提物对脐橙青霉病的防治效果

为开发研制新型植物源柑橘保鲜剂,以抑菌圈直径为指标,利用牛津杯法筛选出对柑橘采后意大利青霉有良好抑制作用的21种中草药的醇提物,并对白薇醇提物防治脐橙青霉病的效果进行了评价。结果表明:白薇、零陵香、鸦胆子、藏青果、白药子5种中草药乙醇提取物对意大利青霉均有较好的抑制效果,抑制圈直径达26 mm以上,其中白薇醇提物抑菌活性最佳,抑制圈直径可达38.39 mm,最小抑菌浓度和最小杀菌浓度分别为3.13 mg/m L和6.25 mg/m L,且抑菌效果与对照药剂1 000倍咪鲜胺无显著差异。25 mg/m L白薇醇提物处理脐橙7 d后,对脐橙青霉病的防治效果达35.93%,与1 000倍咪鲜胺的防治效果相当(36.37%);白薇醇提物处理后可抑制意大利青霉菌菌丝生长,导致菌丝肿胀、膨大、畸形、细胞膜透性增加、细胞内含物外渗;还可诱导脐橙体内过氧化物酶活性下降,而苯丙氨酸解氨酶和几丁质酶活性上升。表明白薇醇提物对脐橙采后青霉病具有较好的防治效果,可进一步开发为植物源柑橘保鲜剂。     

Web Blight of Fan Columbine, <Emphasis Type="Italic">Aquilegia flabellata, </Emphasis>Caused by <Emphasis Type="Italic">Rhizoctonia solani </Emphasis>AG1-IB : A New Disease Record

A new disease, found on fan columbine in August of 1997, first appeared as necrotic spots on leaves and within a week caused wilting of all the leaves. Fungal mycelia bound aerial parts of the plants together, formed mats of mycelia and eventually killed the plants. The pathogen, isolated from the infected leaves and stalks, was identified as Rhizoctonia solani AG1-IB in respect to hyphal anastomosis and culture types. The common name of web blight is proposed for this new occurrence on fan columbine (Kumonosu-byo in Japanese). Received 24 November 1999/ Accepted in revised form 4 February 2000