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     

Alternaria Leaf Spot on Three Species of Pelargonium Caused by Alternaria alternata in Japan

A new disease of pelargonium (Pelargonium domesticum Bailey), ivy geranium (P. peltatum (L.) L'Hér. ex Ait.) and scented geranium (P. graveolens L'Hér.), primarily causing brown spots on leaves, was found in Kawasaki-shi in Kanagawa Prefecture and Tachikawa-shi in Tokyo. An Alternaria sp. was consistently isolated from these diseased leaves, and the isolates were pathogenic to their host leaves. Based on morphological characteristics, the causal fungus in all three cases was identified as Alternaria alternata (Fr.) Keissler. Because Alternaria leaf spot of geranium by A. alternata has already been reported, the pathogenicity of isolates from four groups of genus Pelargonium was investigated. The isolates from scented geranium were pathogenic only to their original host, but the isolates from pelargonium, ivy geranium and geranium were pathogenic to all groups of pelargonium. This is the first report of this disease on pelargonium, ivy geranium and scented geranium caused by A. alternata in Japan. We propose the names for these diseases as Alternaria leaf spot of pelargonium (kappan-byo), Alternaria leaf spot of ivy geranium (kappan-byo) and Alternaria leaf spot of scented geranium (kappan-byo). Received 11 December 2000/ Accepted in revised form 19 July 2001     

Laboratory Evaluation of Citrus Cultivars Susceptibility and Influence of Fruit Size on Fortune Mandarin to Infection by Alternaria alternata pv. citri

Young leaves of 62 citrus cultivars were inoculated with conidia of three Spanish isolates of Alternaria alternata pv. citri, the causal agent of brown spot of citrus. Hybrids with Dancy mandarin, King mandarin or their derivates as a parent, grapefruit cultivars and the mandarin cultivars Guillermina, Emperor, Clemenpons and Esbal were highly susceptible to the pathogen. Satsuma cultivar Clausellina and orange cultivars, with the exception of Sanguinelli, were slightly susceptible. Lemon and lime cultivars were not susceptible, with the exception of Mexican lime (Citrus aurantifolia), which was slightly susceptible. Although this study shows a range of potential hosts for this pathogen, to date the only affected cultivars in Spain are Fortune and Nova mandarins, and Minneola tangelo. The susceptibility of Fortune fruits decreased as diameter increased, being susceptible through the whole season. This was confirmed with field observations in autumn where fruit infections have been detected when the diameter reaches 6–7 cm.     

Alternaria Leaf Spot on Mealycup Sage (<Emphasis Type="Italic">Salvia farinacea </Emphasis>Benth.) Caused by <Emphasis Type="Italic">Alternaria alternata </Emphasis>(Fr.) Keissler

In June 1995, a disease causing round to irregular-shaped, water-soaked, brown to blackish brown spots on mealycup sage (Salvia farinacea Benth.) was found in Atsugi-shi, Kanagawa Prefecture, Japan. The symptoms were seen only on leaves, not on neither flower petals or stems. The disease was also found in Setagaya-ku, Tokyo, Memambetsu-cho, Hokkaido and Shimoda-shi and Matsuzaki-cho, Shizuoka. An Alternaria sp. was frequently isolated from these diseased plants. The isolates were severely pathogenic to mealycup sage and caused lesions on the inoculated leaves. The isolates were also weakly pathogenic on scarlet sage (S. splendens Sellow ex Roem. and Schult.) but not on any other Labiatae plants tested. Based on morphological characteristics, such as size of conidia, chain number, and the short beak on conidia, the causal fungus was identified as Alternaria alternata (Fr.) Keissler. This report is the first on a mealycup sage disease caused by A. alternata. Because the symptom was restricted to the leaf, the common name of Alternaria leaf spot was proposed. Received 30 August 2002/ Accepted in revised form 18 November 2002     

Genetic Analysis of Pathogenicity and Host-specific Toxin Production of Alternaria alternata Tomato Pathotype by Protoplast Fusion

Several pathotypes of Alternaria alternata are known to produce host-specific toxins (HSTs) as agents of pathogenicity or virulence. However, investigations into the genetic controls of HST biosynthesis and pathogenicity of Alternaria pathogens have been limited by the lack of a sexual stage in the life cycle of these pathogens. We report here the development of a protoplast fusion system and its use for genetic analysis of HST production and specific pathogenicity of the tomato pathotype of A. alternata that produces AAL-toxin as a HST. Drug-resistant transformants have been isolated by genetic transformation of nonpathogenic A. alternata (strain O-94) and A. alternata tomato pathotype (strain As-27) with vectors conferring resistance to hygromycin B and geneticin, respectively. Protoplasts of the respective transformants were fused by polyethylene glycol treatment or electrofusion. Fusion products were selected by culturing in the presence of both hygromycin B and geneticin, then confirmed by amplification using a polymerase chain reaction with specific primers to the transforming drug-resistance genes. Stable fusants were purified by successive subcultures on selective medium and single-spore isolation. The resultant stable fusants, probably inter-strain hybrids, had the same pathogenicity and toxin production as the wild-type strain As-27. These results suggest that protoplast fusion has potential applications for genetic analysis of A. alternata pathogens. Received 8 September 2000/ Accepted in revised form 27 October 2000     

Black Spot of Peach Caused by <Emphasis Type="Italic">Alternaria alternata</Emphasis> (Fr.) Keissler

A new disease of peach (Prunus persica Batsch var. vulgaris Maxim.), causing brown, sunken lesions and brownish to blackish brown spots with cracks on peach fruits, was found in Okayama prefecture, Japan, in 1995. The disease was observed not only on peach fruits but also on twigs and leaves. An Alternaria sp. was consistently isolated from these diseased fruits, twigs and leaves. The isolates were pathogenic to peach fruits and leaves. Based on the morphological characteristics, the causal fungus was identified as Alternaria alternata (Fr.) Keissler. After cross-inoculation with isolates from peach, Japanese pear and apple, the isolates were found to be pathogenic only to their original host. This is the first report on a peach disease caused by a host-specific A. alternata; therefore, the common name of black spot (`Kokuhanbyo' in Japanese) was proposed. Received 25 June 1999/ Accepted in revised form 12 October 1999     

Construction and Genetic Analysis of Hybrid Strains between Apple and Tomato Pathotypes of Alternaria alternata by Protoplast Fusion

The genetic controls of host-specific toxin (HST) biosynthesis and the pathogenicity of A. alternata pathogens have been limited by the asexual nature of the life cycle of these fungi. We used a protoplast fusion system for A. alternata to analyze the genetics of HST production and its relation to the specific pathogenicity of these pathogens. Drug-resistant transformants were isolated by genetic transformation, using vectors conferring resistance to hygromycin B and geneticin, for the A. alternata apple pathotype (AM-toxin producer) and A. alternata tomato pathotype (AAL-toxin producer), respectively. Protoplasts of the respective transformants were fused by electrofusion. The majority of resultant stable fusants produced both AM- and AAL-toxins and were pathogenic to susceptible cultivars of both apple and tomato. Pulsed-field gel electrophoresis analysis demonstrated that these fusants (or hybrids) carried small 1.7-and 1.1-Mb chromosomes, characteristic of the parental strains of the apple and tomato pathotypes, respectively. Detection of the AMT gene, involved in AM-toxin biosynthesis, by polymerase chain reaction revealed that all fusants pathogenic to apple maintained this gene. Microfluorimetry analysis using propidium iodide staining suggested that the fusants might be diploid. Received 14 November 2000/ Accepted in revised form 11 December 2000     

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     

Isolation of a Protein Bound to Canker-forming Factor from Citrus Plant

Interaction analysis using affinity analysis (Affinity Sensors, Cambridge, UK) indicated the presence of proteins bound to Apl1, a virulence factor of Xanthomonas campestris pv. citri required for the formation of canker symptom on citrus, in the fraction 25–50% ammonium sulfate of citrus crude extracts. Three proteins of 25, 50 and 110-kD were eluted from an Apl1-affinity column. Western analysis revealed that Apl1 binds specifically to the 25-kD and 110-kD but not to the 50-kD protein. When crude extracts of soybean and of tobacco were applied to the Apl1-affinity column, only faint bands were detected. This result suggests that Apl1 targets exist specifically in citrus plants. The amino acid sequence of the N-terminal of the 25-kD protein was determined, and homology search analysis revealed that this sequence was almost identical to those commonly present in S-adenosyl-l-methionine : trans-caffeoyl-coenzyme A 3-O-methyltransferase (CCoAMT) from several plants. This enzyme is specific to the substrate trans -caffeoyl-CoA and catalyzes the synthesis of trans-feruloyl-CoA for lignin formation. Received 4 November 1999/ Accepted in revised form 26 November 1999     

GhMAPK参与调控棉花对链格孢的抗性

丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)级联途径,由三种级次磷酸化的MAPKKK (MAPK kinase kinase)、MAPKK (MAPK kinase)和MAPK蛋白激酶组成,广泛参与植物防御反应的调控。本文利用已发布的陆地棉Gossypium hirsutum L.全基因组数据,对其中的MAPK家族基因编码序列进行多序列比对和聚类分析。结果显示,53个GhMAPK大都含有植物MAPK保守的TXY基序,并且能够聚集到AtMAPK的A、B、C、D四个族中。进而利用病毒诱导的基因沉默(virus-induced gene silencing, VIGS)技术,研究GhMAPK是否参与调控棉花对链格孢Alternaria alternata抗性。链格孢是引起棉花轮纹斑病并导致棉花早衰的主要病原真菌。结果发现,沉默GhMAPK3、GhMAPK7、GhMAPK9及GhMAPK19的棉花植株,抗病性显著下降,表明这4种GhMAPK正调控棉花对链格孢的抗性。进一步利用靶向性沉默策略,验证这4种GhMAPK中各同源基因对棉花链格孢抗性的调控作用。结果显示,仅同源基因GhMAPK3b、GhMAPK7b、GhMAPK7c、GhMAPK9a、GhMAPK9b、GhMAPK19b和GhMAPK19c参与正调控棉花对链格孢的抗性,而同源基因GhMAPK3a、GhMAPK7a、GhMAPK7d和GhMAPK19a并不调控棉花对链格孢的抗性。     

木霉几丁质酶对烟草赤星病菌的作用

以指形管培养法分别测定几丁质酶粗酶液和纯化的几丁质酶混合液(2种几丁质酶)对烟草赤星病菌孢子萌发的抑制作用。结果表明,较高浓度(25 2U)几丁质酶粗酶液在48h内强烈抑制孢子萌发和芽管伸长,或致芽管畸形和细胞壁破裂;几丁质酶混合液对赤星病菌的孢子萌发也表现出明显的抑制作用,但在相同或相近酶活性条件下,纯化的几丁质酶混合液(9 4U)和粗酶液(12 6U)对赤星病菌孢子萌发的抑制率(处理24h时)分别为46%和84 3%,前者明显低于后者。采用孢子液悬滴法接种烟苗(K 326)叶片测定木霉几丁质酶对赤星病菌致病性的影响。结果表明,几丁质酶粗酶液浓度越高,对孢子萌发抑制时间越长,抑制率越高;其浓度为4 9、9 8、19 5U/ml的抑制率7d时分别为36 8%、56 2%和57 6%。     

Ultrastructural Localization of Hydrogen Peroxide in Host Leaves Treated with AK-toxin I Produced by Alternaria alternata Japanese Pear Pathotype

The rapid generation of reactive oxygen species (ROS), called the oxidative burst, is one of the earliest host responses to pathogen infection or elicitor treatments. Therefore, we looked for the induction of ROS generation in Japanese pear leaves by the host-specific toxin, AK-toxin I using a cytochemical method for detecting H₂O₂. A small amount of non-specific generation of H₂O₂ was found in the cell walls in toxin- and water-treated susceptible and resistant leaves. Thus, the generation of H₂O₂ at cell walls appears to be caused by wounding stress during sampling. Specific generation of ROS, however, was found only in the membrane fragments and extended desmotubules characteristic of modified sites of the plasma membrane in the toxin-treated susceptible leaves. In addition, generation of H₂O₂ at plasma membranes was observed with higher frequency in toxin-treated susceptible leaves. This result indicates that the H₂O₂ generation was associated with damaged sites in the plasmalemma after toxin treatment and perhaps with the formation of membrane fragments from altered portions of the invaginated plasma membrane. Received 21 September 2001/ Accepted in revised form 25 October 2001     

抗菌肽和几丁质酶基因提高烟草对黑胫病菌和赤星病菌的抗性研究

 通过农杆菌介导法将加信号肽修饰的人工合成杂合抗菌肽CEMA基因(SPCEMA),苜蓿防御素基因(AFP),苦瓜几丁质酶基因(CHI)以及SPCEMA-CHI、AFP-CHI、AFP-SPCEMA双价基因导入本明烟(Nicotiana benthamiana),并对转基因烟草T₀和T₁代进行了抗病性检测,比较了不同转基因植株的抗病效果。研究结果表明,转基因烟草对黑胫病菌(Phytophthora parasitica var.nicotianae)、赤星病菌(Alternaria alternata)的抗性均强于非转基因烟草,病情指数差异达极显著水平,其中转AFP-CHI双价基因烟草具有较强的抗性,与单价转基因烟草的抗性差异达显著水平。但各单价转基因以及双价转基因烟草之间对上述病菌并未表现出显著的抗病性差异。结果表明植物源的抗菌肽基因与几丁质酶基因在抗植物真菌病害中具有协同增效作用。     

Alternaria Brown Spot of Minneola in Greece; Evaluation of Citrus Species Susceptibility

During the last three years, a new disease was observed in northwestern Greece on Minneola trees, hybrid of mandarin and grapefruit. On May small brown necrotic leaf spots surrounded by yellow halo areas of various sizes appeared and covered a major portion of the leaves with extension of necrosis into the veins. On young fruits small, slightly depressed black spots were the first symptoms, which later became 2–7 mm in diameter. Brown spots were observed on the leaves and fruits in several orchards in the same area, causing leaves and fruits to drop. In some orchards over 50% of the fruits were affected. From the fruit and leaf spots the typical small-spore species Alternaria alternata was isolated. Pathogenicity tests were performed by artificially inoculating fruits of Minneola, common mandarin and Clementine. The symptoms of the disease were reproduced only on fruits of Minneola hybrids by the specific strain of the fungus Alternaria alternata pv. citri. Different citrus susceptibility tests indicated that mandarins Minneola, Nova and Page were very susceptible to tested isolates while Clementine SRA and Poros Clementine were not. All lemons and lime Seedless were not susceptible. Grapefruit New Hall was not susceptible, while the Star Ruby was. Orange Lane Late, Navel Late, Oval Poros, Olinda, Navel Athos were not susceptible and only Moro showed reaction being slightly susceptible only to one isolate.     

A study on the molecular mechanism of resistance to amicarthiazol in Xanthomonas campestris pv. citri

Three amicarthiazol-resistant mutants (Xuv10, Xuv20 and Xuv40) were obtained by UV induction and used in this study. Minimal inhibition concentrations (MICs) of amicarthiazol against the growth of mutants and wild-type isolate were 400 and 100 microg ml(-1) respectively. Inhibition by amicarthiazol of succinate dehydrogenase (SDH) activities of Xanthomonas campestris pv. citri (Hasse) Dye wild-type isolate (Xcc) and three resistant mutants derived from this isolate were assayed using triphenyltetrazolium chloride (TTC). The SDH activities of these mutants were significantly lower than that of Xcc. The complete nucleotide sequences of four subunits (SdhA, SdhB, SdhC and SdhD) of succinate-ubiquinone oxidoreductase (SQR) were cloned by polymerase chain reaction (PCR) amplification. An amino acid mutation (His229--> Leu229) in sdhB was found to confer resistance of X. campestris pv. citri to amicarthiazol. It is suggested that this mutation alters the SDH complex in some way that prevents binding of amicarthiazol.     

Induced Resistance in Hypocotyl of Cucumber by Infection with Colletotrichum lagenarium in Leaves

Localized infection of cucumber leaves with Colletotrichum lagenarium induced resistance against infection after challenge inoculation with pathogenic fungi, including C. lagenarium and Pythium ultimum var. ultimum. Systemic acquired resistance in the hypocotyl was observed when challenge inoculation was made 4 to 7 days after the first inoculation of cotyledons. Seven days after the first inoculation of a primary leaf, induced resistance against the challenge inoculation in the hypocotyl was also observed. However, the hypocotyl did not develop induced resistance when plants were challenged by a wound inoculation with P. ultimum. Received 9 June 1999/ Accepted in revised form 13 December 1999