Antagonistic effects of soil bacteria onFusarium oxysporum Schlecht f. sp.dianthi (Prill and Del.) Snyd. and Hans

Summary Fusarium oxysporum f. sp.dianthi, pathogenic on carnation plants is very sensitive toBacillus subtilis M51 inhibition.Fusarium oxysporum disease (fusariosis) is prevented for a period of two months after treatment of plants withBacillus subtilis M51. The persistence ofB. subtilis M51, marked for selenomycin resistance (MZ51) and inoculated on the roots of carnation cuttings was studied. Soil used was two types: naturally infested withFusarium oxysporum and free from this pathogen. Bacterial cells presence on the roots was detected by direct plating and the presence of the pathogen in the roots was investigated by histological assays. Evidence gathered by these procedures suggest that plant protection is dependent on the physical presence ofB. subtilis M51 cells on the roots.     

Effects of arbuscular mycorrhiza (AM) on health ofLinum usitatissimum L. infected by fungal pathogens

Effects of arbuscular mycorrhizal (AM) symbiosis on health ofLinum usitatissimum infected by fungal pathogens were investigated exemplarily. Physiological and biochemical analyses were done to explain the mechanisms underlying the AM effects. AM plants showed increased resistance against the wilt pathogen (Fusarium oxysporum f. sp.lini), the level of this effects depended on the plant cultivars which all showed the same level of root colonization by arbuscular mycorrhizal fungi (AMF). In contrary to that, AM plants were highly susceptible against the shoot pathogenOidium lini, but they suffered less than non-AM plants in terms of shoot fresh weight, CO₂ assimilation and content of sucrose in shoot apex. This indicates that AM not only activates resistance mechanisms but also can induce tolerance against pathogens. The concentration of phytohormones such as auxin- and gibberellin-like substances were increased in shoots of AM plants. In roots the ethylene production was increased, too. Furthermore the content and composition of free sterols were highly altered in leaves of AM plants. Root infection by AMF caused an increased respiratory activity and a reduced degree of DNA methylation, but both modifications only occurred in infected root parts indicating an increasing gene activity. The presented results suggest that nearly all parts of a plant are influenced by AM but not in the same manner. In the case of mildewed linseed the effect of AM on plant health was impressing, it indicates that AM has an ability to induce tolerance.     

Colonization of cucumber plants by the biocontrol fungus Clonostachys rosea f. catenulata

Clonostachys rosea f. catenulata (Gliocladium catenulatum) strain J1446 (formulated as Prestop WP) suppressed Fusarium root and stem rot caused by Fusarium oxysporum f. sp. radicis-cucumerinum (Forc) on cucumber plants grown hydroponically in rockwool medium. Sixty days following application at seeding, the biocontrol agent had proliferated through the rockwool blocks and was present on cucumber roots and the crown region of the stem at populations >1 × 10⁵ CFU/g fresh weight. Scanning electron micrographs showed that C. rosea had rapidly colonized the root surface and was associated with root hairs and epidermal cell junctions. Following transformation of the fungus with Agrobacterium tumefaciens strain AGL-1 containing the hygromycin resistance (hph) and β-glucuronidase (uidA) genes, blue-stained mycelia could be seen growing on the surface and within epidermal and cortical cells of roots, stems and shoots 3 weeks after treatment. Quantification of GUS activity by fluorometric assays showed that fungal biomass was highest in the roots and crown area, while the extent of colonization of upper stems and true leaves was variable. Higher population levels resulted following application to rockwool blocks compared to seed treatment. Application of C. rosea preceding inoculation with Forc significantly reduced pathogen populations on roots compared to plants inoculated with Forc alone. Colonization of infection sites in the root zone reduced pathogen development and disease incidence. Densities of the biocontrol agent appeared to increase in the presence of the pathogen.     

Stratospheric Ozone Depletion: High Arctic Tundra Plant Growth on Svalbard is not Affected by Enhanced UV-B after 7 years of UV-B Supplementation in the Field

The response of tundra plants to enhanced UV-B radiation simulating 15 and 30% ozone depletion was studied at two high arctic sites (Isdammen and Adventdalen, 78° N, Svalbard).The set-up of the UV-B supplementation systems is described, consisting of large and small UV lamp arrays, installed in 1996 and 2002. After 7 years of exposure to enhanced UV-B radiation, plant cover, density, morphological (leaf fresh and dry weight, leaf thickness, leaf area, reproductive and ecophysiological parameters leaf UV-B absorbance, leaf phenolic content, leaf water content) were not affected by enhanced UV-B radiation. DNA damage in the leaves was not increased with enhanced UV-B in Salix polaris and Cassiope tetragona. DNA damage in Salix polaris leaves was higher than in leaves of C. tetragona. The length of male gametophyte moss plants of Polytrichum hyperboreum was reduced with elevated UV-B as well as the number of Pedicularis hirsuta plants per plot, but the inflorescence length of Bistorta vivipara was not significantly affected. We discuss the possible causes of tolerance of tundra plants to UV-B (absence of response to enhanced UV-B) in terms of methodology (supplementation versus exclusion), ecophysiological adaptations to UV-B and the biogeographical history of polar plants     

In vitro selection of alfalfa plants resistant to Fusarium oxysporum f. sp. medicaginis

Summary From two lines of Medicago sativa characterized by a high regeneration capability, calli resistant to culture filtrate of Fusarium oxysporum f. sp. medicaginis have been selected. In these calli regeneration capability was greatly reduced and only one plant per callus was recovered. Regenerated plants have been evaluated for resistance to culture filtrate and for in vivo resistance to the pathogen. Three plants out of eight were resistant to the fungus and a high correlation between resistance to culture filtrate and in vivo resistance was observed.Research work supported by C.N.R., Italy. Special grant I.P.R.A. Subproject 1, paper no. 1468     

Evaluation of the leaf juice of some higher plants for their toxicity against soil borne pathogens

Out of the leaf juices of eighteen plant species screened, only Eupatorium cannabinum exhibited complete toxicity against Pythium debaryanum, Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsii. Shade drying of the leaves had no adverse effect, while oven drying produced an adverse effect on the fungitoxicity of the leaves of E. cannabinum. The crude leaf juice of E. cannabinum successfully inhibited damping-off (Fusarium oxysporum) infection of Pisum sativum seedlings.     

嫁接西瓜对西瓜枯萎病菌侵染的防御机制研究

为了揭示嫁接提高西瓜抗枯萎病的机制,该研究以嫁接西瓜为材料,采用扫描电镜观察了枯萎病菌侵染下寄主的组织结构变化,荧光定量分析了相关防卫基因的表达,比较了嫁接西瓜对枯萎病菌侵染的抗感反应。结果显示:(1)枯萎病菌侵染后,与自根西瓜相比,嫁接西瓜的根部木质部导管通过快速形成膜状物、侵填体及细胞壁增厚阻塞菌丝入侵;自根西瓜防御反应较嫁接西瓜晚,严重侵染时薄壁细胞降解,导管组织脱落导致维管系统空洞,从而使植株呈现萎蔫症状,该现象在嫁接西瓜中没有发现。(2)枯萎病菌侵染后,嫁接西瓜比自根西瓜具有较高的防卫基因表达水平,其中:嫁接西瓜中,CHI、APX和PPO基因的表达随枯萎病菌侵染时间的延长而升高,而PAL呈现先升高后降低的表达趋势,但仍高于本底表达;自根西瓜中,仅PPO基因在枯萎病菌侵染后表达上调,而其他基因的表达则是先升高后降低,与嫁接西瓜中的PAL基因表达一致。研究表明,嫁接植株一方面通过快速的组织结构响应,另一方面从转录水平提高了相关防卫基因的表达,最终使植株具有抗病性;推测防御基因在嫁接植株与枯萎病菌互作中的强烈诱导响应可能是嫁接植株抗病的分子机制之一。     

Reduction of ambient UV-B radiation does not affect growth but may change the flowering pattern of Rosmarinus officinalis L.

The effects of sub-ambient levels of UV-B radiation on the shrub Rosmarinus officinalis L. were investigated in a field filtration experiment in which the ambient UV-B was manipulated by a combination of UV-B transmitting and UV-B absorbing filters. As a result, the plants were receiving near-ambient or drastically reduced UV-B radiation doses. Drastic reduction of UV-B radiation had no effect on mean, total and maximum stem length, number of stems per plant, dry mass of leaves, stems and roots and leaf nitrogen and phenolic contents. However, flowering was more pronounced under reduced UV-B radiation during the winter period which coincides with ascending ambient UV-B radiation. In contrast, during autumn and early winter, a period which coincides with descending ambient UV-B radiation, flowering was unaffected by reduced UV-B radiation. We can conclude that natural UV-B radiation does not affect growth of Rosmarinus officinalis, but its reduction could influence the flowering pattern of the species.     

Interaction of Fusarium Oxysporum f.sp. Cubense with Pseudomonas Fluorescens Precolonized to Banana Roots

Effect of precolonization of banana cv Neeypovan roots with Pseudomonas fluorescens on infection with Fusarium oxysporum f.sp. cubense was studied. Under in vitro conditions Pseudomonas fluorescens clearly inhibited Fusarium oxysporum f.sp. cubense. Fluorescein isothiocyanate-tagged antibodies raised in a rabbit system for Pseudomonas fluorescens and Fusarium oxysporum f.sp. cubense separately were used to study the spread of both organisms in banana root. It was observed that precolonization with Pseudomonas fluorescens could reduce Fusarium oxysporum f.sp. cubense colonization by 72%, and also correlated with a number of structural changes in the cortical cells, mainly with densely stained amorphous material and polymorphic wall thickenings as revealed by light and electron microscopic studies. Massive depositions of unusual structures at sites of fungal entry was also noticed, which clearly indicated that bacterized root cells were signalled to mobilize a number of defence structures for preventing the spread of pathogen in the tissue. This revised version was published online in July 2006 with corrections to the Cover Date.     

香蕉幼苗三类有机小分子溶质对尖孢镰刀菌侵染的生理响应

为阐明香蕉枯萎病发病机制,研究了尖孢镰刀菌侵染后,香蕉植株中几种对尖孢镰刀菌生长有显著作用的物质(氨基酸、有机羧酸、酚酸)种类和含量的变化。结果表明:(1)病原菌侵染后,伤害逐渐加剧,株高和生物量显著下降。(2)病原菌侵染后,叶片氨基酸总量显著升高,其中丝氨酸、缬氨酸、组氨酸、异亮氨酸和亮氨酸增幅较大,病原菌侵染16 d,其含量分别为侵染前的7.1、6.2、4.4、3.5和2.3倍;而根氨基酸总量开始显著降低,差异逐渐变小。(3)叶片有机羧酸酸含量在病原菌侵染后显著增加,而在根中显著降低。侵染植株叶片中草酸、柠檬酸、苹果酸、琥珀酸和延胡索酸含量分别是未侵染植株叶片的2.6、1.6、1.9、1.8和2.3倍;根中草酸、柠檬酸、苹果酸、琥珀酸和延胡索酸含量分别是未侵染植株的81%、42%、44%、28%和59%。(4)病原菌侵染后,植株叶片和根中酚酸含量都显著升高。叶片中阿魏酸、肉桂酸和水杨酸含量分别是未侵染叶片的2.9、1.7和2.9倍;而根中对羟基苯甲酸和丁香酸含量分别是未侵染根的4.3和1.5倍。研究结果表明,尖孢镰刀菌侵染后,植物与病原菌的相互作用使得植物体内抑菌物质和促菌物质都会相应的增加,植株对病害有一定的抗性,但促菌物质种类和含量较高最终使得感病植株发病。     

Colonization of root tissues and protection against Fusarium wilt of rye (Secale cereale) by nonpathogenic rhizosphere strains of Fusarium culmorum

Colonization of rye (Secale cereale) tissues by nonpathogenic rhizosphere Fusarium culmorum isolates DEMFc2 and DEMFc5 and a pathogenic strain DEMFc37, and their effect on plant fresh weight were studied in pot experiments. Both rhizosphere isolates colonized the epidermis and the cortex but were not found in vessels, while the pathogen colonized all three layers of root cells. The numbers of pathogen CFU isolated from plant tissues were much higher than those of the rhizosphere isolates in spite of the same number of macroconidia used as inoculum (1 × 10⁵ g⁻¹ of soil). Inoculation of seedlings with DEMFc2 resulted in a 20% increase, with DEMFc5 in more than a 20% reduction, and with DEMFc37 in a 38% reduction of shoot fresh weight of 14-day-old plants. Pre-colonization of plants with (either of) the rhizosphere isolates and subsequent inoculation with the pathogen resulted in plant weights the same as those observed in plants inoculated with the rhizosphere strain alone. The disease severity index for shoots of plants pre-colonized with DEMFc2 was reduced from class 4 (86% diseased plants) observed for plants inoculated with the pathogen alone to class 2 (average of 8% diseased plants) when pre-treated with the rhizosphere strain. The CFU number of the pathogen isolated from the interior of roots of plants pre-colonized with the rhizosphere isolates was as low as 10% of the number isolated from plants inoculated with the pathogen alone. A study of in vitro interactions between the rhizosphere isolates and the pathogen suggests that changes in plant colonization by the pathogen and its effect on fresh weight of plants pre-colonized with the rhizosphere isolates were not connected with inhibition of its growth by a direct action of the rhizosphere isolates. The results suggest that strain DEMFc2 can be considered as a potential biocontrol agent.     

Fungus gnats vector Fusarium oxysporum f.sp. radicislycopersici1

Fungus gnat adults transported Fusarium oxysporum f.sp. radicis-lycopersici from Petri dish culture and infected host plants to the roots and hypocotyls of healthy tomato and bean plants. The source of the fungus did not affect the ability of fungus gnats to transport the fungus to healthy hosts. The presence of fungus gnat larvae in media in which young tomato plants were grown did not increase the incidence of plant infection by the pathogen. Fungus gnat adults appear to aid in the dissemination of F. oxysporum f.sp. radicis-lycopersici.     

Agrobacterium rhizogens mediated transformation of Rauvolfia serpentina: Regeneration and alkaloid synthesis

Shoot cultures of Rauvolfia serpentina infected with Agrobacterium rhizogenes 15834 produced tumourous tissue at the site of injection, which eventually developed callus with hairy roots. Sporadic shoot formation occurred from the hairy roots. The shoots were grown to maturity in the green house. The mature transformed plants (RST) showed distinct variations in their physiological characteristics. The flowers of the transformed plants were more delicate and less pigmented when compared to the flowers of the mature normal plants. The roots of the transformed plants were hairy with a number of lateral branches, whereas the roots of the normal plant had very few lateral branches. The biomass of the transformed plant was 86.39 g/plant (fresh wt), significantly higher than the normal plant which was 77.3 g/plant (fresh wt). The total alkaloid content in the mature transformed plant (0.073 g per plant) was similar to the normal plant (0.078 g per plant), although the hairy roots contained little alkaloid.Abbreviations MS Murashige & Skoog's basal medium - MLS modified Linsmaier & Skoog's basal medium - BA benzyladenine - NAA naphthalene acetic acid - TLC thin layer chromatography - HPLC high performance liquid chromatography     

Molecular characterization of pathogenic Fusarium species in cucurbit plants from Kermanshah province, Iran

Fusarium is one of the important phytopathogenic genera of microfungi causing serious losses on cucurbit plants in Kermanshah province, the largest area of cucurbits plantation in Iran. Therefore, the objectives in this study were to isolate and identify disease-causing Fusarium spp. from infected cucurbit plants, to ascertain their pathogenicity, and to determine their phylogenetic relationships. A total of 100 Fusarium isolates were obtained from diseased cucurbit plants collected from fields in different geographic regions in Kermanshah province, Iran. According to morphological characters, all isolates were identified as Fusarium oxysporum, Fusarium proliferatum, Fusarium equiseti, Fusarium semitectum and Fusarium solani. All isolates of the five Fusarium spp. were evaluated for their pathogenicity on healthy cucumber (Cucumis sativus) and honeydew melon (Cucumis melo) seedlings in the glasshouse. F. oxysporum caused damping-off in 20–35 days on both cucurbit seedlings tested. Typical stem rot symptoms were observed within 15 days after inoculation with F. solani on both seedlings. Based on the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) restriction fragment length polymorphism (RFLP) analysis, the five Fusarium species were divided into two major groups. In particular, isolates belonging to the F. solani species complex (FSSC) were separated into two RFLP types. Grouping among Fusarium strains derived from restriction analysis was in agreement with criteria used in morphological classification. Therefore, the PCR-ITS-RFLP method provides a simple and rapid procedure for the differentiation of Fusarium strains at species level. This is the first report on identification and pathogenicity of major plant pathogenic Fusarium spp. causing root and stem rot on cucurbits in Iran.     

Effect of nitrogen supply rate on disease resistance in tomato depends on the pathogen

The aim of this study was to investigate the effect of tissue nitrogen concentration, as a consequence of nitrogen supply rate, on the susceptibility of tomato plants to three pathogens. We varied tissue N concentration by supplying N at different rates by adding nitrate in different, exponentially increasing amounts to the nutrient solution on which the tomato plants were grown. Separate experiments were carried out to test susceptibility of tomato plants to the bacterial speck-causing Pseudomonas syringae pv tomato, to the wilt agent Fusarium oxysporum f.sp. lycopersici and to tomato powdery mildew caused by Oidium lycopersicum. The effect of tissue N concentration appeared to be highly pathogen-dependent: there was no effect on susceptibility to F. oxysporum, but susceptibility to P. syringae and O. lycopersicum increased significantly with increasing N concentration. We have previously demonstrated the opposite for susceptibility to Botrytis cinerea: decreasing susceptibility with increasing N concentration. The apparent contradictory effects are discussed in relation to the effect of N supply on both the nutritional value of the plant tissue to the pathogen and on the concentration of resistance-related compounds. We conclude that the effect of changing both characteristics on disease susceptibility is highly pathogen-specific and is probably dependent on differences in resource requirements of the pathogen or the sensitivity of the pathogen to plant resistance reactions or on both these factors. This revised version was published online in August 2006 with corrections to the Cover Date.     

Increased photosyntethic efficiency generated by fungal symbiosis in Agave victoria-reginae

A unique non-pathogenic strain of Fusarium oxysporum was isolated from field-collected plants of Agave victoria-reginae Moore. It was then re-introduced into micropropagated plants of the same species under greenhouse conditions. The fungic inoculation induced a 225% increase in the length of roots, 50% in the number of root branches and 50% in the number of stomata on the adaxial surface of leaves. Also, an increment of 167% in nocturnal acidity and a 122% in malic acid was observed and nocturnal pH was significantly more acid in the inoculated plants. Total chlorophyll and sugar content increased 14 and 172%, respectively. These results indicate a higher photosynthetic efficiency of the plants inoculated with the fungus than those plants which were not inoculated; therefore the association of this unique F. oxysporum strain with A. victoria-reginae Moore was considered as a beneficial symbiosis.     

Piriformospora indica,a plant-root-colonising fungus enhances growth and tolerance of wheat to seedlings damping-off caused by Fusarium oxysporum

In greenhouse experiments, we investigated the potentials of Piriformospora indica (Pi) to penetrate and colonise roots of wheat and to induce beneficial effects on growth as well as to reduce seedlings damping-off disease of wheat caused by the soil-borne fungus Fusarium oxysporum (Fo). By microscopy we observed the intracellular hyphae within root cortex cells of 7–14 day-old plants and chlamydospores within root cortex cells of 14–28 day-old plants and within root hair cells of 21–28 day-old plants. Moreover, diagnostic PCR based on the β-tubulin gene marker confirmed the presence of Pi in roots of inoculated plants. Also, we found that plants inoculated with Pi exhibited a better growth of roots and shoots as well as early flowering as compared with non-treated plants. Moreover, Pi conferred tolerance of wheat to seedlings damping-off i.e. by reducing the harmful effects of Fo on infected plants.     

The efficiency of two new formulated biofungicides in the control of damping-off and root rot of cucumber and improving the plant defence system

The ability of Rhizoleen-T and Rhizoleen-B to suppress the roots’ diseases of cucumber caused by Fusarium oxysporum and Rhizoctonia solani and induction of the plant defence system was evaluated. The results showed a significant suppression in damping-off and root rot of cucumber when the two biofungicides were applied. They increased the surviving percentage of the treated plants to 98.0% and reduced the mean disease rating to 1.0. The biofungicides significantly enhanced the plant’s morphology and physiology when compared with either infected or non-infected control. The mechanism of action of the biofungicides is could be through the enhancement of the plant defences, in addition to their antifungal effect. They stimulated the defence of the plants by increasing the content of proline and phenols and induction the defensin genes. Biofungicides induced defence genes in the 50-day-old treated plants. The biofungicides are effective, cost effective and applicable in the control of root diseases of cucumber.     

香蕉植株根区土壤的真菌多样性分析

尖孢镰孢菌古巴专化型(Fusarium oxysporum f.sp.cubense)是香蕉枯萎病的病原菌,该菌是一种土壤习居菌,了解香蕉根区土壤中真菌多样性及镰孢菌属(Fusarium)真菌所占比例,对如何减少土壤中的病原菌、预防香蕉枯萎病的发生有重要的指导意义。该文通过采集不同宿根年限的香蕉健康植株和枯萎病植株的根区土壤,利用高通量测序技术测定土壤样品中的真菌种群。结果表明:(1)同一宿根年限的香蕉植株中,健康植株根区土壤中所获的reads及OTUs数量均高于枯萎病植株,说明健康植株根区土壤的真菌多样性丰富于枯萎病植株。(2)除了一年生香蕉枯萎病植株以担子菌门(Basidiomycota)为主外,其他土壤样品中均以子囊菌门(Ascomycota)为主,其中的丛赤壳科最高相对丰度来自三年生健康植株的根区土壤(26.02%),其次是五年生的枯萎病植株根区土壤(15.56%)。(3)在丛赤壳科中,镰孢菌属在三年生健康植株土壤中的相对丰度最高(2.54%),在其他样品中的相对丰度在0.1%～0.65%之间;在镰孢菌属中,腐皮镰孢菌(Fusarium solani)的相对丰度(0～1.59%之间)高于尖孢镰孢菌(F.oxysporum),尖孢镰孢菌仅占很小的比例(相对丰度0～0.08%之间)。可见,在不同香蕉植株的根区土壤中,健康植株的根区土壤真菌多样性高于枯萎病植株,无论是健康植株还是枯萎病植株的根区土壤中,作为香蕉枯萎病病原菌的镰孢菌属或尖孢镰孢菌的群体均不占主导地位。     

Increased solar UV-B radiation may reduce infection by arbuscular mycorrhizal fungi (AMF) in dune grassland plants: evidence from five years of field exposure

An area of coastal dune grassland, dominated by the gramineous species Calamagrostis epigeios and Carex arenaria, was exposed to enhanced levels of UV-B radiation during a five year period. These species showed reduced AM-fungal infection percentages in their roots. In C. epigeios AM infection was reduced by 18%, C. arenaria showed a reduction by 20%. The major effect of enhanced UV-B on AM associations was a reduction of the number of arbuscules. This indicates a reduction in the exchange of nutrients between the symbionts. Since the effect of UV-B on AM associations may result from altered flavonoid levels in the root exudates of the host plants, flavonoid levels in the roots were investigated. No detectable flavonoid concentrations were found in the roots of C. epigeios and C. arenaria. Less effective AM associations can have pronounced negative effects on biodiversity and nutrient dynamics of the dune grassland ecosystem. The possible mechanisms causing these indirect effects of elevated UV-B on below ground AM associations are discussed. We conclude that UV-B induced changes in plant hormone levels are more likely to be the mechanism reducing AMF infection than UV-B induced alterations in flavonoid concentrations in the root exudates of the host plant.