Multiplication of isolate R-5 of <Emphasis Type="Italic">Streptomyces galbus</Emphasis> on rhododendron leaves and its production of cell wall-degrading enzymes

 An endophytic actinomycete, isolate R-5 of Streptomyces galbus Frommer, that has promising potential as a biocontrol agent was originally isolated from field-grown rhododendron. In this study, the mode of entry of R-5 into leaves of tissue-cultured seedlings of rhododendron was investigated in connection with its production of cell wall-degrading enzymes. Light and scanning electron microscopy (SEM) revealed that R-5 grew on leaf surfaces and entered leaf tissues via stomata and that the internal mycelia grew out of stomata after colonization in host tissues. Micromanipulation at the SEM level demonstrated a prominent depression in the host surface at the interfaces with the mycelia, suggesting that such a depression could be caused by degradation of cell wall components by hydrolytic enzymes secreted from R-5 mycelia. In subsequent plate assays, R-5 produced cellulase, pectinase, xylanase, and nonspecific esterase when cultured in liquid medium. Moreover, R-5 multiplied in mineral medium containing cellulose, pectin, or xylan as a single carbon source. Thus, R-5 mycelia could degrade host cell walls at contact sites and probably utilize the degradation products as carbon sources. Received: May 16, 2002 / Accepted: July 9, 2002     

Studies on Endophytic Actinomycetes ( I ) <Emphasis Type="Italic">Streptomyces </Emphasis>sp. Isolated from Rhododendron and Its Antifungal Activity

To survey endophytic actinomycetes as potential biocontrol agents against fungal diseases of rhododendron, young plants of rhododendron were surface-sterilized for use as an isolation source. Nine, six and two isolates, with distinguishing characteristics based on the macroscopic appearance of colonies, were obtained from roots, stems and leaves, respectively, suggesting that various species of actinomycetes grow in the respective organs of this plant as symbionts or parasites. On an agar medium, only isolate R-5 commonly formed a clear growth-inhibition zone against two major fungal pathogens of rhododendron, Phytophthora cinnamomi and Pestalotiopsis sydowiana, indicating that this isolate can produce antifungal material(s). Acetone extracts of a liquid culture of R-5 had a broad antimicrobial spectrum against Gram-positive bacteria, yeast and filamentous fungi. Isolate R-5 was identified as a Streptomyces sp. based on morphological, physiological and chemotaxonomical characteristics. The present results indicate that isolate R-5 is a suitable candidate for the biocontrol of diseases of rhododendron. Received 25 March 2000/ Accepted in revised form 18 May 2000     

Identification of endophytic <Emphasis Type="Italic">Streptomyces</Emphasis> sp. R-5 and analysis of its antimicrobial metabolites

In a previous study, endophytic Streptomyces sp. R-5 isolated from a field-grown rhododendron was able to confer disease resistance on tissue-cultured seedlings of rhododendron when applied to medium surfaces in flasks. Here, the isolate was identified as Streptomyces galbus Frommer based on various physiological characteristics and analysis of the 16S rDNA sequence. Its major antimicro-bial metabolites were identified as actinomycin X₂ and fungichromin by analyses using liquid chromatography/mass spectometry and nuclear magnetic resonance.     

Disease resistance induced by nonantagonistic endophytic Streptomyces spp. on tissue-cultured seedlings of rhododendron

Of 82 strains of endophytic actinomycetes isolated from rhododendron plants, 12 were not antagonistic against Pestalotiopsis sydowiana, which is the causal agent of Pestalotia disease. Of these 12, MBR-37 and MBR-38 (identified as Streptomyces spp.) grew on IMA-2 medium. Tissue-cultured seedlings of rhododendron treated with these nonantagonistic strains showed less wilting and/or smaller lesions to P. sydowiana, although the degree of resistance was a little lower than that conferred by antagonistic Streptomyces galbus strain R-5. These seedlings accumulated the anthocyanin(s), suggesting that resistance induced by these strains could depend on activated defense responses associated with the phenylpropanoid pathway rather than with antibiosis.     

Potential for eradication of the exotic plant pathogens Phytophthora kernoviae and Phytophthora ramorum during composting

Temperature and exposure time effects on Phytophthora kernoviae and Phytophthora ramorum viability were examined in flasks of compost and in a large‐scale composting system containing plant waste. Cellophane, rhododendron leaf and peat‐based inoculum of P. kernoviae and P. ramorum isolates were used in flasks; naturally infected leaves were inserted into a large‐scale system. Exposures of 5 and 10 days respectively at a mean temperature of 35°C in flask and large‐scale composts reduced P. kernoviae and P. ramorum inocula to below detection limits using semi‐selective culturing. Although P. ramorum was undetectable after a 1‐day exposure of inoculum to compost at 40°C in flasks, it survived on leaves exposed to a mean temperature of 40·9°C for 5 days in a large‐scale composting system. No survival of P. ramorum was detected after exposure of infected leaves for 5 days to a mean temperature of ≥41·9°C (32·8°C for P. kernoviae) or for 10 days at ≥31·8°C (25·9°C for Phytophthora pseudosyringae on infected bilberry stems) in large‐scale systems. Fitted survival probabilities of P. ramorum on infected leaves exposed in a large‐scale system for 5 days at 45°C or for 10 days at 35°C were <3%, for an average initial infection level of leaves of 59·2%. RNA quantification to measure viability was shown to be unreliable in environments that favour RNA preservation: high levels of ITS1 RNA were recovered from P. kernoviae‐ and P. ramorum‐infected leaves exposed to composting plant wastes at >53°C, when all culture results were negative.     

玉米灰斑病研究方法：Ⅰ玉米灰斑病菌孢子的培养

为了明确玉米灰斑病菌的产孢条件,将玉米灰斑病菌接种到马铃薯蔗糖（PSA）、燕麦番茄碳酸钙（OTCA）、玉米叶粉碳酸钙（MLPCA）、燕麦粉（OA）、粗面粉(CWPA)等5种培养基上,于25 ℃下培养,测定该菌生长速度、产孢时间和产孢量。结果表明,菌丝在上述培养基上生长速度均较慢,每天平均生长0.33 mm左右,其中以在OA和PSA上生长速度相对较快。基于上述结果,构建了玉米灰斑病菌孢子培养的二级分段培养法,即将菌丝块接种于加有10～15粒直径为5 mm玻璃珠的马铃薯蔗糖（PS）液体培养基三角瓶里,在25 ℃,180 r/min条件下振荡培养15 d,形成菌丝悬液,再取菌丝悬浮液涂布在MLPCA 培养基上,避光培养13 d左右,可获得大量供接种的分生孢子。长期光照促进菌丝生长,不利于孢子的形成,而长期黑暗不利于菌丝生长,但有利于孢子的形成。     

A Monitoring System for Green Fluorescence Protein Gene-transformed Fusarium oxysporum in Melon Seedlings

Using melon seedlings at the cotyledon stage and genetically marked fungi, a system for monitoring pathogenic and nonpathogenic Fusarium oxysporum was devised in the present study. Protoplasts were prepared from three formae speciales (melonis, radicis-lycopersici and fragariae )of F. oxysporum and transformed with a synthetic gene for green fluorescence protein. Transformants were primarily isolated in the presence of hygromycin B and then screened by the emission of bright green fluorescence. Roots of melon seedlings were inoculated with fluorescing microconidia of these fungi, and fungal infection behavior was traced. Using fluorescence microscopy, we directly observed not only the fungus at the root surface, but also the mycelia elongating in the trachea of roots. Both pathogenic and nonpathogenic fungi germinated and hyphae elongated superficially on the surface of root. Only pathogenic fungi caused root necrosis at the inoculation site. Hyphae grew within the stem to induce constriction or cracking of lower hypocotyls, then causing wilting of the seedlings. Infection behavior of genetically marked pathogenic and nonpathogenic F. oxysporum could be successfully monitored after inoculation of cotyledons of seedlings. Received 6 June 2001/ Accepted in revised form 3 August 2001     

Induction of systemic resistance byPseudomonas fluorescens Pf1 againstXanthomonas oryzae pv.Oryzae in rice leaves

When lower leaves of rice plants were inoculated with powder formulation of a saprophytic strain ofPseudomonas fluorescens, Pfl, upper leaves, in addition to the inoculated lower leaves, showed resistance to the rice bacterial blight pathogenXanthomonas oryzae pv.oryzae. When the leaves were challenge-inoculated withX. oryzae pv.oryzae 4 days afterP. fluorescens application on lower leaves, the disease intensity in upper leaves decreased from 6.7 to 1.1. When rice seeds were treated with the formulation ofP. fluorescens Pfl and sown, 30-day-old seedlings showed resistance toX. oryzae pv.oryzae and the disease intensity decreased from 6.8 to 1.2. The induced resistance was transient; leaves sprayed withP. fluorescens Pfl at 30 days after treatment and leaves of 60-day-old seedlings fromP. fluorescens-treated seeds did not show resistance to the pathogen. In field trials, seed treatment followed by foliar application of the powder formulation ofP. fluorescens Pfl effectively controlled rice bacterial blight and increased the yield. In the induced resistant leaves a sharp increase in lignification and activities of peroxidase, phenylalanine ammonia-lyase and 4-coumarate: CoA ligase was observed when the leaves were challenge-inoculated withX. oryzae pv.oryzae. An approximately threefold increase in lignin content, peroxidase activity and phenylalanine ammonia-lyase activity and a fivefold increase in 4-coumarate: CoA ligase activity were observed 5 days after challenge inoculation withX. oryzae pv.oryzae in rice leaves pretreated withP. fluorescens for 5 days. A similar increase in defense-related activities was not observed in susceptible interactions or inP. fluorescens-treated plants at later stages of interactions when no resistance to the pathogen was observed.     

Ralstonia solanacearum preferential colonization in the shoot apical meristem explains its pathogenicity pattern in tomato seedlings

Ralstonia solanacearum causes a lethal bacterial wilt disease in many plants by colonizing the vascular tissues of the hosts. Upon inoculation of tomato seedlings through either leaf or root, the wilting symptoms occur first at the apical region and then proceed downward along the shoot. The systemic order of the disease initiation and progression in the host, independent of the site of pathogen inoculation, is yet to be investigated. To understand the disease progression more clearly, we have carried out a systematic study of the pathogen localization by GUS staining of inoculated tomato seedlings, at 24-hour intervals from 0 days post-inoculation (dpi) to 5 dpi. In both inoculation methods, pathogen colonization was observed at 1 dpi at the apical meristem as well as the cotyledon leaves, where the disease initiates. As the disease progressed, colonization by the pathogen towards the lower region of the shoot was observed. Disease consistency and pathogenicity magnitude were observed to be higher using the leaf inoculation method than the root inoculation method. Several R. solanacearum transposon-induced mutants that were reduced in virulence by root inoculation but virulent by leaf inoculation were obtained. Using GUS staining, it was observed that these mutants were unable to localize in the shoot region when inoculated in the root. Our study indicates that the apical meristem and the cotyledon leaves are the first regions to be colonized in inoculated tomato seedlings, which might explain the disease initiation from this region.     

非亲和性稻瘟病菌对水稻的诱导抗性

为研究稻瘟病菌Magnaporthe oryzae不同菌株间的相互作用,选择与单抗性基因系水稻IRBL5-M （携带抗性基因Pi5）表现为亲和性的菌株HN52与非亲和性的菌株HN119为研究对象,将其单独或混合接种到单抗性基因系水稻IRBL5-M中,并通过荧光显微镜观察接种后水稻叶鞘的发病情况及病斑面积,测定接种后水稻内相关抗性基因OsWRKY45、OsNPR1、OsPR10、OsMAPK2的表达量以及活性氧的变化。结果显示,相较于单独接种亲和性菌株,混合接种后单抗性基因系水稻IRBL5-M病斑发病面积减少;混合接种中亲和性菌株HN52菌丝侵染能力降低,侵染菌丝细胞间扩展率显著降低73.13%;同时单抗性基因系水稻IRBL5-M中OsWRKY45、OsNPR1、OsPR10和OsMAPK2抗性基因表达量显著增加,水稻叶片中活性氧含量增加,表明在菌株混合侵染过程中,非亲和性菌株可通过激发水稻的抗性反应来降低亲和性菌株对水稻的侵染程度。     

Black Rot of Artichoke Leaves Caused by Two Phoma Species in Japan

Phoma macrostoma var. incolorata and P. exigua var. exigua were isolated from leaf spots of artichoke in Mie Prefecture, Japan in June 1997. Colonies of P. macrostoma var. incolorata grew fast, pigmented the medium vinous without staining the mycelium, and changed blue with NaOH. Conidial size in culture was 3 to 10 μm × 2 to 4.5 μm. Colonies of P. exigua var. exigua grew fast without pigmentation and changed yellow-green with NaOH. The conidia were sometimes septate, and size of aseptate conidia was 4 to 9.5 μm × 2 to 3.5 μm. These fungi produced gray, round spots on artichoke leaves. This report is the first on the occurrence of artichoke black rot caused by P. macrostoma var. incolorata and P. exigua var. exigua in Japan. Received 5 July 2001/ Accepted in revised form 12 March 2002     

Brown Leaf Spot on Lantana spp. Caused by Pseudocercospora guianensis

Brown leaf spot of Lantana camara L. and L. montevidensis Briq. caused by Pseudocercospora guianensis (Stevens et Solheim) Deighton was found in Shizuoka, Chiba, Kagoshima and Okinawa Prefectures. Pathogenicity of isolates from the leaf spots was examined, and a taxonomic study as well as identification of the causal fungus was carried out. Similar leaf spots appeared on 7 to 25 days after inoculation with the isolates on Lantana spp., and the same fungus was re-isolated from the inoculated leaves. Two hitherto known Pseudocercospora species on Lantana, P. guianensis and P. formosana, were considered to be variations within one species, and we identified the causal fungus as P. guianensis due to priority. Received 25 September 2000/ Accepted in revised form 20 May 2001     

植物根围促生细菌菌剂增强玉米干旱耐受性研究

选用植物根围促生细菌(PGPR)菌剂BBS处理玉米幼苗,研究干旱胁迫下玉米幼苗叶片相关生理特性及基因表达差异。结果表明,干旱胁迫15 d后,BBS处理组玉米幼苗的萎蔫程度显著低于对照组;处理组植株叶片MDA含量仅为对照组的74.35%,叶片脯氨酸和可溶性糖含量分别是对照组的3.39倍和1.07倍;处理组植株叶片ZmP5CS1的表达水平及SOD活性均显著高于对照组;BBS处理组植株叶片在干旱胁迫过程中一直保持较低的H_2O_2含量。研究得出,BBS处理能够有效减缓干旱胁迫对玉米幼苗造成的伤害,进而加速正常浇水后的恢复。处理组植株叶片NCED1和ZmDREB2.7的高水平表达也表明干旱胁迫下玉米幼苗与BBS的互作过程可能有依赖ABA信号途径及不依赖ABA信号途径的共同参与。     

Impact of Foliar Diseases on Photosynthesis, Protein Content and Seed Yield of Alfalfa and Efficacy of Fungicide Application

Foliar pathogens attack alfalfa wherever the crop is grown, but their impact, especially on seed production, is poorly understood. In greenhouse trials, leaf spot injury caused by inoculation with various pathogens reduced the crude protein content of infected alfalfa leaves by 22% compared with a healthy control. There was a negative relationship between disease injury and the photosynthetic efficiency of alfalfa plants, as determined by measuring chlorophyll fluorescence in leaves from inoculated vs. non-inoculated seedlings. In field trials at two sites in Alberta from 2001 to 2003, inoculation with Phoma medicaginis increased disease incidence in four of six trials, Phoma sclerotioides increased incidence in four of five trials, and Leptosphaerulina trifolii and Stemphylium botryosum increased incidence in two of six trials. There was a trend for inoculation treatments to reduce seed yield, despite high levels of background infection by indigenous pathogens. The fungicides benomyl and propiconazole inhibited radial growth of Phoma spp. in vitro and reduced disease incidence in inoculated greenhouse experiments. In field trials, applications of benomyl and propiconazole reduced disease incidence, but did not always increase seed yield.     

Root Rot of Miniature Roses Caused by Pythium helicoides

Miniature roses growing in an ebb-and-flow watering system developed dieback during the summer growing season of 1996 in Gifu Prefecture. The main diagnostic symptoms were chlorosis of leaf followed by blight, and a brown, water-soaked root rot followed by dieback. Pythium isolates were recovered from the rotted root. The isolates form proliferous ellipsoidal papillate sporangia, spherical smooth oogonia, elongate antheridia, and aplerotic oospores. The optimum temperature for hyphal growth was 35°C with a growth rate of 34 mm/24 hr. Optimum temperature of zoospore formation (25-30°C) was lower than that of mycelial growth, and zoospores were produced even at 10°C. The isolates were identified as P. helicoides on the basis of these characteristics. In pathogenicity tests disease severity was highest at the highest tested temperature (35°C) at which the disease naturally occurred in summer. Four days after inoculation, the leaves turned yellow and the roots had a water-soaked rot, followed by leaf blight and root dieback after 7 days. The disease transmission test showed that diseased plants were found throughout the bench after 10 days. Received 4 July 2001/ Accepted in revised form 10 October 2001     

Identification of sugar beet germplasm EL51 as a source of resistance to post-emergence Rhizoctonia damping-off

Resistance of sugar beet seedlings to Rhizoctonia damping-off caused by Rhizoctonia solani has not been described. A series of preliminary characterisations using a single susceptible host and four different R. solani isolates suggested the disease progression pattern was predictable. Two AG-4 isolates and a less virulent AG-2-2 isolate (W22) showed a comparable pattern of disease progression in the growth chamber where disease index values increased for the first 5–6 days, were relatively constant for the next 7–8 days, and declined thereafter. Seedlings inoculated with a highly virulent AG-2-2 isolate (R-1) under the same conditions showed similar patterns for the first 4 days post-inoculation; however disease index values continued to increase until seedling death at 13–14 days. Similar results were observed in the greenhouse, and a small expanded set of other germplasm lines were screened. One tested germplasm accession, EL51, survived seedling inoculation with R. solani AG-2-2 R-1, and its disease progress pattern was characterised. In a field seedling disease nursery artificially inoculated with R. solani AG-2-2 R-1, seedling persistence was high with EL51, but not with a susceptible hybrid. Identification of EL51 as a source of resistance to Rhizoctonia damping-off may allow investigations into the Beta vulgaris–Rhizoctonia solani pathosystem and add value in sugar beet breeding.     

Antagonistic Pythium against pathogenic Pythium on cucumber roots1

Antagonistic microorganisms introduced into the growing medium, the seed or the root, have been shown to reduce the attack of many soil-borne plant pathogenic fungi. In 1983, three experiments with Pythium oligandrum were carried out using cucumber seedlings artificially inoculated with Pythium splendens. The seedlings cv. Ideal Nova were grown in shallow boxes containing granulated rockwool fibre (Grodania). The plants were grown at a minimum temperature of 20°C in a glasshouse. Plants were treated with the antagonist either by coating the seeds, or by applying a disc of a PDA-culture to 9-day old seedlings. The pathogen was introduced 9 or 5 days later, respectively, by adding PDA culture discs to the rockwool; 15 and 38 days later, the area with attacked roots was calculated as a percentage of the box-bottom area. The experiments showed that it is possible to control P. splendens by either method of application. P. oligandrum was able to colonize the seed and rhizosphere and suppress P. splendens for at least 38 days. If future experiments are similarly positive, the methods for use in commercial horticulture should be worked out as soon as possible.     

Effect of Figaren, an Antiviral Protein from Cucumis figarei, on Cucumber mosaic virus Infection

Local lesion formation on cowpea leaves was more than 50% inhibited by treatment with a 23 kDa RNase-like glycoprotein from Cucumis figarei, figaren, from 24 hr before to 1 hr after inoculation with Cucumber mosaic virus (CMV). CMV accumulation detected by ELISA in tobacco leaves treated with figaren 6 or 0 hr before inoculation with CMV was suppressed. When upper leaves of tobacco plants were treated with figaren and inoculated 10 min later with CMV, mosaic symptoms were delayed for 5–7 days on most of the tobacco plants, and some plants remained asymptomatic. From fluorescence in situ hybridization, infection sites were present in figaren-treated cowpea or melon leaves after inoculation with CMV, though the sites were reduced in number and size compared with those in water-treated control leaves. The amount of CMV RNAs and CMV antigen in melon protoplasts inoculated with CMV and subsequently incubated with figaren similarly increased with time as did that in the control. ELISA and local lesion assays indicated that CMV infection on the upper surfaces of the leaves of tobacco, melon, cowpea and C. amaranticolor whose lower surfaces had been treated with figaren 5–10 min before CMV inoculation was almost completely inhibited. Figaren did not inhibit CMV infection on the opposite untreated leaf halves of melon, cowpea and C. amaranticolor, whereas it almost completely inhibited CMV infection on the untreated halves of leaves of tobacco. CMV infection was not inhibited in the untreated upper or lower leaves of the four plants. These data suggest that figaren does not completely prevent CMV invasion but does inhibit the initial infection processes. It may also induce localized acquired resistance in host plants. Received 10 October 2000/ Accepted in revised form 6 February 2001     

Development of Oculimacula yallundae and O. acuformis (eyespot) on leaf sheaths of winter wheat in the UK in relation to thermal time

In a controlled environment (15/10°C) (day/night) container experiment on winter wheat (cv. Avalon), eyespot incidence (percentage of plants affected) and number of leaf sheaths penetrated after 6 weeks increased with inoculum concentration (10²−10⁶ conidia mL⁻¹) of Oculimacula yallundae (OY) or Oculimacula acuformis (OA), but there was no difference between the two species. In an outdoor container experiment, seedlings inoculated with OY 2 weeks after sowing had a greater incidence of eyespot than those inoculated with OA, when assessed 7 weeks after inoculation. Seedlings inoculated with OA at 10 or 20 weeks after sowing developed more severe eyespot by maturity than those inoculated with OY. In an experiment at 15/10°C with seedlings inoculated with OY + OA 2 weeks after sowing, more leaf sheaths were penetrated by OY (3·0 per plant) than OA (2·3 per plant) 6 weeks after inoculation. Field experiments with winter wheat consistently showed leaf sheath production, leaf sheath death, and number of leaf sheaths infected or penetrated by OA or OY were related linearly to thermal time (°C days) after sowing. Depending on cultivar, season and sample, a new leaf sheath was produced in 116–216°C days; a leaf sheath died in 221–350°C days; and infection of a new leaf sheath occurred in 129–389°C days. The mean number of living leaf sheaths infected differed between samples, cultivars and seasons for both OY and OA. Regression analysis of the 1985/86 data suggested that OY progressed more rapidly than OA through the leaf sheaths, and that both the pathogens progressed more rapidly than the rate of leaf sheath death, but more slowly than the rate at which leaf sheaths were produced. It also suggested that OA progressed more slowly than the rate at which leaf sheaths died in 1987/88, but OY did not.     

Induced systemic resistance of Chinese cabbage to bacterial leaf spot and <Emphasis Type="Italic">Alternaria </Emphasis>leaf spot by the root endophytic fungus, <Emphasis Type="Italic">Heteroconium chaetospira</Emphasis>

 The root endophytic fungus Heteroconium chaetospira isolate OGR-3 was tested for its ability to induce systemic resistance in Chinese cabbage against bacterial leaf spot caused by Pseudomonas syringae pv. maculicola and Alternaria leaf spot caused by Alternaria brassicae of the foliar diseases. Chinese cabbage seedlings planted in soil infested with an isolate of H. chaetospira were incubated in a growth chamber for 32 days. The first to fourth true leaves of the seedlings were challenge-inoculated with P. syringae pv. maculicola or A. brassicae. Chinese cabbage planted in soil infested with H. chaetospira showed significant decreases in the number of lesions of bacterial leaf spot or Alternaria leaf spot when compared to the control plants not treated with H. chaetospira. The results indicated that colonization of roots by H. chaetospira could induce systemic resistance in Chinese cabbage and reduce the incidence of bacterial leaf spot and Alternaria leaf spot. Received: April 24, 2002 / Accepted: August 9, 2002