Cultivar‐dependent yield reductions in white clover due to simulated grazing and White clover mosaic virus infection are associated with differences in storage reserves and antioxidant metabolism

White clover is considered amongst the most important forage legumes in temperate agricultural systems. It contributes to increased quality of pastures through its ability to fix atmospheric nitrogen, providing high nutritional feed for livestock. White clover is host to the potexvirus White clover mosaic virus (WClMV), which is readily transmitted via animal trampling/grazing and mowing. WClMV infection may strongly impact white clover's performance, affecting its ability to compete with other plants in the pasture. Understanding the extent to which the virus influences white clover's growth and yield may contribute to improve pastoral management by pointing out the importance of stress factors on pasture plant performance and herbage yield. In the current study, the effects of WClMV infection and simulated grazing on white clover's fitness, oxidative damage and the induction of antioxidants were investigated. Two cultivars of white clover, Grasslands Huia and Grasslands Kopu II, were subjected to three harvests. In response to WClMV infection and grazing‐like cutting, white clover experienced oxidative stress as evidenced by the accumulation of lipid peroxides and protein carbonyls. Biomass accumulation, given as fresh weight and dry matter content, was also negatively affected. Both stresses induced an increase in ascorbate and glutathione levels and enhanced the activity of superoxide dismutase, ascorbate peroxidase, glutathione peroxidase, catalase and glutathione reductase. Glutathione S‐transferase activity and the levels of reduced ascorbate and reduced glutathione were not affected by either stress.     

小麦抗条锈反应中活性氧及细胞质钙离子的作用

 为探讨活性氧和细胞质钙离子在小麦抗条锈病反应中的作用,以小麦品种洛夫林13与具有不同致病力的单孢锈菌CY29、CY25的互作体系为平台,对锈菌侵染后小麦叶片中活性氧(ROS)积累、保护酶系(SOD、CAT和APX)活性变化动态、细胞质膜的透性改变及细胞质钙离子浓度变化做了研究。结果表明,不亲和锈菌CY25侵染可引起小麦叶片内2次ROS的爆发,第1次出现在接种后前期(接种后第2天),强度较小,第2次出现在接种后期(接种后第5天),强度较大;亲和锈菌CY29侵染只引起1次ROS爆发,出现在接种后期(接种后第5和第6天之间),但强度极大。过敏性坏死反应HR只出现在不亲和互作小麦叶片上前期ROS爆发之后,表明前期ROS爆发与HR的产生有关。伴随着后期小麦叶片中强度极高的ROS的爆发,叶片细胞原生质膜遭到了破坏,细胞内物质外渗,细胞不久便死亡,表明高强度的ROS爆发会导致细胞死亡。根据不同互作体系ROS爆发时期SOD、CAT和APX等保护酶的活性变化分析,不亲和互作体系前期强度较小的ROS爆发主要成分是H₂O₂,后期强度较高的ROS爆发主要成分是O₂^-_·和H₂O₂;亲和互作体系强度极高的ROS爆发主要成分是O₂^-_·和H₂O₂。由此说明H₂O₂是引起小麦抗病反应HR发生的因素,而O₂^-_·则是引起细胞死亡的因素。细胞质钙离子浓度变化研究表明,HR的发生与细胞质钙离子浓度增加相关。细胞质钙离子浓度的降低推迟了HR的发生,这说明小麦叶片细胞内细胞质钙离子浓度的增加是HR的必要条件,同时也说明Ca²⁺是植物HR的胞内第二信使参与植物抗病防卫反应。     

The Relationship of Host Range, Physiology, and Genotype to Virulence on Cantaloupe in Pseudomonas syringae from Cantaloupe Blight Epidemics in France

ABSTRACT In 1993, a bacterial blight caused important losses of cantaloupe (Cucumis melo var. cantalupensis) in southwestern France and has now been reported in all cantaloupe-growing regions of France. The causal agent of this blight is Pseudomonas syringae, although on a worldwide basis this bacterium has not been a major pathogen of melon for over 50 years. To identify the pathovar of the cantaloupe pathogen, we employed biochemical tests, plasmid and chromosomal profiling, and host range studies for 23 strains from cantaloupe and 47 reference strains of 14 pathovars of P. syringae. Numerical analysis of 119 traits, serological typing, syringomycin production, and BOX-polymerase chain reaction profiles did not allow us to differentiate among pathovars related to P. syringae pv. syringae. Host range studies of cantaloupe and references strains on 18 plant species showed that virulence to sugar beet was a common feature of strains virulent on cantaloupe, but was not common to strains avirulent on cantaloupe. Virulence to other species of plants varied among strains, but the overall extent of the host range was proportional to aggressiveness to cantaloupe. We propose that the strains attacking cantaloupe in France be considered P. syringae pv. aptata and that adequate host range testing may reveal that this pathovar is the cause of cantaloupe blight reported in other parts of the world.     

Activation of systemic disease resistance in pea by an avirulent bacterium or a benzothiadiazole, but not by a fungal leaf spot pathogen

Inoculation of first expanded leaves of pea seedlings with an avirulent strain of Pseudomonas syringae pv. pisi , or treatment with sprays of a benzothiadiazole (20 or 100  μ g a.i. mL⁻¹), decreased the susceptibility of subsequent leaves 7 or 14 days later to challenge inoculation with Mycosphaerella pinodes . Inoculation of first leaves with a virulent strain of P. syringae pv. pisi or with M. pinodes did not decrease the susceptibility of plants to M. pinodes . Treatments effective in decreasing susceptibility to M. pinodes were similarly active against Uromyces viciae-fabae and virulent P. syringae pv. pisi . Effective treatments also enhanced the activities of the enzymes β-1,3-glucanase and chitinase in untreated upper leaves 6 days later. Ineffective treatments for decreased susceptibility had no effect on the activity of the enzymes. None of the treatments enhanced peroxidase activities. The results are discussed in relation to the reported signalling effects of the benzothiadiazole and in relation to a suggested high activity of the avirulent P. syringae pv. pisi strain and inactivity of M. pinodes in enhancing natural signalling.     

ROS and NO production in compatible and incompatible tomato-<Emphasis Type="Italic">Meloidogyne incognita</Emphasis> interactions

Nitric oxide (NO) has been shown to be an essential regulatory molecule in plant response to pathogen infection in synergy with reactive oxygen species (ROS). At the present, nothing is known about the role of NO in disease resistance to nematode infection. We used a resistant tomato cultivar with different sensitivity to avirulent and virulent populations of the root-knot nematode Meloidogyne incognita to investigate the key components involved in oxidative and nitrosative metabolism. We analyzed the superoxide radical production, hydrogen peroxide content, and nitric oxide synthase (NOS)-like and nitrate reductase activities, as potential sources of NO. A rapid NO accumulation and ROS production were found at 12 h after infection in compatible and incompatible tomato-nematode interactions, whereas the amount of NO and ROS gave different results 24 and 48 h after infection amongst compatible and incompatible interactions. NOS-like arginine-dependent enzyme rather than nitrate reductase was the main source of NO production, and NOS-like activity increased substantially in the incompatible interaction. We can envisage a functional overlap of both NO and ROS in tomato defence response to nematode invasion, NO and H₂O₂ cooperating in triggering hypersensitive cell death. Therefore, NO and ROS are key molecules which may help to orchestrate events following nematode challenge, and which may influence the host cellular metabolism.     

<Emphasis Type="Italic">Trichoderma harzianum</Emphasis>- mediated reprogramming of oxidative stress response in root apoplast of sunflower enhances defence against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Trichoderma harzianum is an effective biocontrol agent against the devastating plant pathogen Rhizoctonia solani. Despite its wide application in agriculture, the mechanisms of biocontrol are not yet fully understood. Mycoparasitism and antibiosis are suggested, but may not be sole cause of disease reduction. In the present study, we investigated the role of oxidant-antioxidant metabolites in the root apoplast of sunflower challenged by R. solani in the presence/absence of T. harzianum NBRI-1055. Analysis of oxidative stress response revealed a reduction in hydroxyl radical concentration (^•OH; 3.6 times) at 9 days after pathogen inoculation (dapi), superoxide anion radical concentration (O₂^•−; 4.1 times) at 8 dapi and hydrogen peroxide concentration (H₂O₂; 2.7 times) levels at 7 dapi in plants treated with spent maize-cob formulation of T. harzianum NBRI-1055 (MCFT), as compared to pathogen-inoculated plants. The protection afforded by the biocontrol agent was associated with the accumulation of the ROS gene network: the catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and ascorbate peroxidase (APx), maximum activity of CAT (11.0 times) was observed at 8 dapi, SOD (7.0 times) at 7 dapi, GPx (5.4 times) and APx (8.1 times) at 7 dapi in MCFT-treated plants challenged with the pathogen. This was further supported by the inhibition of lipid and protein oxidation in Trichoderma-inoculated plants. MCFT stimulated the accumulation of secondary metabolites of phenolic nature that increased up to five-fold and also exhibited strong antioxidant activity at 8 dapi, eventually leading to the systemic accumulation of phytoalexins. These results suggest that T. harzianum–mediated biocontrol may be related to alleviating R. solani-induced oxidative stress.     

青枯菌(Pseudomonas solanacearum)在番茄抗、感病品种根部的吸附、侵入和繁殖

 采用不同的方法接种和利用扫描、透射电镜观察,研究不同致病力的青枯菌对番茄抗病及感病品种根部的吸附、侵入与繁殖。发现番茄抗病品种与感病品种的植株体内在菌体数量上有明显差异,而与青枯菌对番茄根部的吸附关系不显著。电镜观察发现青枯菌强致病力菌株菌体能以游离的形式存在于番茄感病品种根部的细胞间隙中,并能降解植株细胞壁、破坏原生质膜;青枯菌强致病力菌株菌体在抗病品种根内和青枯菌强致病力菌株在抗病及感病品种根内均被番茄植株细胞壁吸附,并且被细胞壁周围的浓密物质所包围。     

Temporal modulation of oxidant and antioxidative responses in Brassica carinata during β-aminobutyric acid-induced resistance against Alternaria brassicae

A preinoculative foliar application of 5 mM BABA significantly inhibited the colonization of Alternaria brassicae on leaves of Brassica carinata susceptible cultivar car6. BABA treatment led to transient but significant increase in H₂O₂ level during early stages of pathogen colonization. A significant increase in superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol dependent peroxidase (GDP) contributed to inhibition of the oxidative stress in BABA treated plants in response to pathogen infection. In conclusion BABA treatment led to proper balance of oxidant & antioxidants suitable for expression of resistance resulting in curtail of pathogen ingress during early stages of colonization.     

烟草野火病菌(Pseudomonas syringae pv. tabaci)诊断试剂盒的制备及应用

 本研究通过筛选致病力强的烟草野火病菌株作为抗原,制备了烟草野火病菌特异性的抗血清,研制出SPA-ELISA、间接-ELISA 2种诊断试剂盒,使检测真正作到了简便、快速、灵敏、准确。同时,应用这2种诊断试剂盒对从田间及温室中采集的土壤、烟株根围、田间杂草、种子进行检测,明确了云南烟草野火病的初侵染源主要为种子和根围。     

Durability of Resistance and Cost of Virulence

A seasonal model, where a growing season is defined as the time between sowing and harvest and alternates with an inter-crop period, was derived to study the effects of the ‘cost of virulence’ and cropping ratio on durability of resistance. We assumed a single strain of virulent pathogen, a single strain of avirulent pathogen and two cultivars (one resistant and one susceptible) and studied two measures of durability of resistance (‘take-over time’ and ‘usefulness time’). Take-over time is defined as the time needed for the virulent strain of the pathogen to reach a preset threshold and predominate over the previous pathogen population. Usefulness time is the time needed before the estimated gain in green canopy area duration per plant through the use of the resistant cultivar becomes negligible. The model suggested that, although it could take several seasons before the virulent strain of the pathogen predominated over the previous pathogen population, the usefulness time of the resistant cultivar was always much shorter. Furthermore, increasing selection for the virulent strain of the pathogen (through increasing the cropping ratio of the resistant cultivar) caused the virulent strain of the pathogen to invade the system more rapidly. Cost of virulence, reflecting differences in pathogen infection rates between the four possible combinations of cultivar/pathogen strain, significantly affected durability of resistance, with the dynamics of the virulent and avirulent strains ranging from a case where the virulent strain of the pathogen died out to a case where the virulent strain of the pathogen invaded the resident pathogen population. An intermediate state, where the system reached equilibrium and the virulent strain of the pathogen neither became predominant nor died out, was defined as ‘coexistence’ of both strains of the pathogen. Occurrence of coexistence was directly related to the cost of virulence since it did not occur when virulence of the pathogen did not have a fitness cost. Two methods to include cost of virulence in the model gave similar results in relation to the two measures of durability of resistance studied.     

Inhibition by bacterial lipopolysaccharide of wilt induced by phytopathogenic bacteria in cut cherry shoots, and of cherry canker disease

Virulent phytopathogenic bacteria and their less virulent mutants induced wilt in cut cherry shoots treated with cell suspensions. Both heterologous and homologous bacteria induced the will reaction. The wilt-inducing isolates also caused the hypersensitive response in tobacco. An avirulent capsulaled mutant of Erwinia amylovora did not elicit either response. Non-phytopathogens did not cause wilting to occur. All the tested isolates aggregated after entry into plant tissues whether wilting occurred or not, but did not aggregate in suspensions in contact with cut shoots. Bacterial lipopolysaccharide or sidechain carbohydrate derived from lipopolysaccharide of Pseudomonas syringae pv. morsprunorum protected shoots against wilt when administered with the same bacterial isolate. These substances also inhibited cherry canker disease in susceptible mature trees when applied before inoculation to leaf-scar inoculation sites. Inhibition of bacterial adhesion to mature plant tissues treated with lipopolysaccharide or sidechain is suggested as a cause of disease inhibition. The wilt reaction may provide a rapid test of phytopathogenicity.     

Pseudomonas syringae pv. avellanae pathovar nov., the bacterium causing canker disease on Corylus avellana

The results of an extensive study of 170 morphological, physiological, biochemical, serological and pathological characters of 18 representative isolates from the filbert canker pathogen are presented. These indicate that the bacterium responsible for the disease of filberts ( Corylus avellana ) in Greece is properly characterized as a typical member of the species Pseudomonas syringae. It is placed in group Ia according to the LOPAT tests. The differences in some characters between Pseudomonas syringae pv. syringae and the filbert Pseudomonas , as well as its host specificity, justify its classification as a new pathovar of Pseudomonas syringae and the name of Pseudomonas syringae pv. avellanae pv.nov. is proposed for the bacterium causing the disease 'bacterial canker' of Corylus avellana.     

Overexpression of phytoglobin in barley alters both compatible and incompatible interactions with the mildew pathogen Blumeria graminis

This study showed how barley plants can be shifted in their response to isolates of the mildew pathogen Blumeria graminis with different host adaptation by overexpression of the barley phytoglobin gene HvHb1. At early infection stages, plants overexpressing phytoglobin (GPHb1) showed less papilla formation and more hypersensitive response against both virulent and avirulent pathogen isolates compared to the wildtype (WT) plants. The shift was most pronounced in a wheat-adapted isolate (B. graminis f. sp. tritici). At later infection stages, GPHb1 plants infected with a virulent pathogen isolate (A6) showed less leaf chlorosis compared to the WT plants, indicating delayed senescence. The chlorophyll level was significantly higher in A6-infected GPHb1 plants 9 days after inoculation (dai) and the senescence indicators sphingosine-1-phosphate:ceramide ratio and phytol content confirmed delayed senescence. At 14 dai the percentage of fungal DNA was significantly higher on the GPHb1 plants than on WT plants, probably as a result of the delayed senescence. The results show that overexpression of phytoglobin (previously known as plant haemoglobin) can be an important tool to understand disease-related stress effects in plants of agronomic importance and for understanding basic resistance mechanisms. Studying this process in more detail may provide insights into how to alleviate stress-related senescence in plants.     

Biological Control of Pseudomonas syringae pv. syringae, the Causal Agent of Basal Kernel Blight of Barley, by Antagonistic Pantoea agglomerans

ABSTRACT Strains of Pantoea agglomerans (synanamorph Erwinia herbicola) suppressed the development of basal kernel blight of barley, caused by Pseudomonas syringae pv. syringae, when applied to heads prior to the Pseudomonas syringae pv. syringae infection window at the soft dough stage of kernel development. Field experiments in 1994 and 1995 revealed 45 to 74% kernel blight disease reduction, whereas glasshouse studies resulted in 50 to 100% disease control depending on the isolate used and barley cultivar screened. The efficacy of biocontrol strains was affected by time and rate of application. Percentage of kernels infected decreased significantly when P. agglomerans was applied before pathogen inoculation, but not when coinoculated. A single P. agglomerans application 3 days prior to the pathogen inoculation was sufficient to provide control since populations of about 10(7) CFU per kernel were established consistently, while Pseudomonas syringae pv. syringae populations dropped 100-fold to 2.0 x 10(4) CFU per kernel. An application to the flag leaf at EC 49 (before heading) also reduced kernel infection percentages significantly. Basal blight decreased with increasing concentrations (10(3) to 10(7) CFU/ml) of P. agglomerans, with 10(7) CFU/ml providing the best control. For long-term preservation and marketability, the survival of bacterial antagonists in several wettable powder formulations was tested. Over all formulations tested, the survival declined between 10- to >100-fold over a period of 1.5 years (r = -0.7; P = 0.000). Although not significant, storage of most formulations at 4 degrees C was better for viability (90 to 93% survival) than was storage at 22 degrees C (73 to 79%). However, long-term preservation had no adverse effect on biocontrol efficacy.     

Occurrence of bacterial brown spot of dry beans in the Transvaal province of South Africa

Bacterial brown spot, caused by Pseudomonas syringae pv. syringae , caused epidemics on dry beans ( Phaseolus vulgaris ) on the Transvaal Highveld in South Africa during 1992. Crop losses of up to 55% were reported in plantings of the dry bean cultivar Bonus. The pathogen was detected in commercial seed stocks.     

Production of syringomycins and syringopeptins by Pseudomonas syringae pv. atrofaciens

All virulent strains of Pseudomonas syringae pv. atrofaciens produce in vitro substances with syringomycin-like features. All strains inhibited the growth of Geotrichum candidum in the plate assay although the extent of their growth inhibition was variable.
Purification of bioactive culture extracts of a highly virulent strain by ion exchange chromatography (Whatman CM52) yielded a main fraction which inhibited the growth of Rhodotorula pilimanae and Bacillus megaterium , and was phytotoxic to tobacco and wheat plants. In particular, the injection of this fraction in the culm of wheat plants caused phytotoxic symptoms on leaves similar to those caused in nature by the pathogen. The further purification by HPLC of the above fraction gave rise to four main bioactive substances which have been identified by spectroscopic methods (FAB-MS) and amino acid analysis as syringomycin E, syringomycin G, syringopeptin 25A and syringopeptin 25B, toxic lipodepsipeptides thus far recognized to be produced by most strains of P. syringae pv. syringae . The injection of both syringomycin E and syringopeptin 25A in wheat leaves caused necrotic symptoms; however, syringopeptin 25A was at least six times more active than syringomycin E. The possible role of the toxins in the disease development on cereals and the need for a careful examination of pathogenetic and biochemical features of P. syringae pv. atrofaciens to establish the relationships of the two pathovars in the 'syringae group' are discussed.     

Distinguishing pathovars of Pseudomonas syringae on peas: nutritional, pathogenicity and serological tests

Of the published methods to distinguish Pseudomonas syringae pv. syringae and Pseudomonas syringae pv. pisi , inoculation of susceptible cultivars was the most reliable. Results were confirmed by inoculation of lemon fruit.
A much more rapid and convenient serological method was developed to distinguish the two pathovars. Antisera against glutaraldehyde-fixed cells had a high level of specificity in Ouchterlony gel double-diffusion tests and, after cross-absorption with heterologous antigens, in indirect ELISA. Antiserum to P. syringae pv. pisi has considerable potential to detect pea seed infected with this pathogen.     

Histochemical studies on the accumulation of reactive oxygen species (O2 and H2O2) in the incompatible and compatible interaction of wheat—Puccinia striiformis f. sp. tritici

The generation and accumulation of reactive oxygen species (ROS), superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂), were studied in the interaction between wheat cv. ‘Suwon 11’ and two races of Puccinia striiformis f. sp. tritici (avirulent and virulent). Generation of O₂⁻ and H₂O₂ was analyzed histochemically using nitroblue tetrazolium (NBT) and 3,3-diamino-benzidine (DAB), respectively. At the pre-penetration stage during appressorium formation both stripe rust races induced H₂O₂ accumulation in guard cells. In the incompatible interaction, a rapid increase of O₂⁻ and H₂O₂ generation at infection sites was detected. The percentage of infection sites showing NBT and DAB staining was 36.1% and 40.0%, respectively, 12 h after inoculation (hai). At extended incubation time until 24 hai, percentage of infection sites showing H₂O₂ accumulation further increased, whereas those exhibiting O₂⁻ accumulation declined. The early infection stage from 12 to 24 hai coincided with primary haustoria formation in mesophyll cells. In contrast, in the compatible interaction, O₂⁻ and H₂O₂ generation could not be detected in most of the infection sites. In the incompatible interaction, intensive DAB staining was also determined in mesophyll cells, especially in cell walls, surrounding the infected cells 16–24 hai; thereafter, these cells contained fluorescing compounds and underwent hypersensitive response (HR). The number of necrotic host cells surrounding the infection sites increased continuously from 20 to 96 hai. It might be concluded that H₂O₂ accumulation during the early infection stage is associated with the occurrence of hypersensitive cell death and that resistance response is leading to arrest the avirulent race of the obligate stripe rust pathogen. In the compatible interaction at 96 hai, H₂O₂ accumulation was observed in mesophyll cells surrounding the rust lesion.