Identification of bacterial protein markers and enolase as a plant response protein in the infection of Olea europaea subsp. europaea by Pseudomonas savastanoi pv. savastanoi

Olive knot disease is characterised by the development of galls on Olea europaea stems as a result of infection by Pseudomonas savastanoi pv. savastanoi. Protein differential accumulation during the first week of infection was studied using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry to investigate the biochemical changes occurring in infected tissues and to understand the factors involved in bacteria pathogenesis and plant response to infection. Common infection symptoms were obtained in 1 year-old plants of two Portuguese cultivars, ‘Galega’ and ‘Cordovil de Serpa’ using the strain NCPPB 2327. The comparison of protein patterns of non-inoculated stem tissues, stem tissues inoculated with water or with the strain NCPPB 2327 led to the detection of differentially expressed infection-related proteins. Moreover a distinct protein pattern was obtained between cultivars in response to infection. The differential protein expression was characterised by qualitative and quantitative variation. Among the differentially expressed proteins were the bacterial P. savastanoi orthologues of outer membrane porin F, the tellurium resistance protein, aconitate hydratase 2 and a hypothetical protein with unknown function. From O. europaea, protein orthologues of enolase and calcium-dependent protein kinase were found to be differentially expressed. Results are discussed in the context of the molecular basis of plant–pathogen interactions in the search for markers for the presence of the bacterium in plant tissues.     

Imazethapyr inhibition of acetolactate synthase in Rhizobium and its symbiosis with pea

Acetolactate synthase (ALS) activity extracted from Rhizobium leguminosarum biovar. viciae has been characterized. The optimum pH for extraction was 7·6 and for the assay 7·0. The K_m for pyruvate was 7·2 mM , and the enzyme was saturated at 40 mM . An obligatory requirement of TPP and Mg²⁺ for full ALS activity was observed. Valine was the only branched-chain amino acid that caused ALS feedback inhibition. The specific activity of Rhizobium ALS was nearly 20 times the activity found in pea (Pisum sativum) leaves. Bacteroids from pea nodules also showed high ALS activity, and the nodule plant fraction had higher ALS activity than other plant tissues. ALS sensitivity to imazethapyr was also dependent on the source: ALS activity of free-living Rhizobium and bacteroids was slightly more tolerant than that of other pea tissues, but the differences were less than those found in rates of specific activity. It is proposed that the high ALS activity expressed by Rhizobium, both as free-living bacteria and as bacteroids, is related to the growth tolerance of rhizobia to imazethapyr and is also related to the relative tolerance of symbiotic pea plants. © 1998 SCI     

Accumulation of Pathogenesis-Related Proteins in the Apoplast of a Susceptible Cultivar of Apple (Malus domestica cv. Elstar) after Infection by Venturia inaequalis and Constitutive Expression of PR Genes in the Resistant Cultivar Remo

Leaves of apple (Malus domestica cv. Elstar) were infected with a cloned isolate of the apple scab Venturia inaequalis. The intercellular washing fluid (IWF) of these plants was collected and the variation in the composition of proteins in the IWF was analysed by SDS-PAGE and two-dimensional gel electrophoresis during and after the infection with V. inaequalis, the causal agent of apple scab. The subsequent analysis of induced proteins by electron spray ionization quadrupole time of flight mass spectroscopy revealed the presence of -1,3-glucanase, chitinase, thaumatin-like protein and a cysteine-like protease in M. domestica leaves infected by V. inaequalis. These results were confirmed by immunoblotting with antibodies against some of these proteins. Moreover, a non-specific lipid transfer protein was identified in uninfected leaves: the amount declined to a non-detectable level within the first week after infection by V. inaequalis. The analysis of the IWF of M. domestica cv. Remo, bearing resistances to apple scab, powdery mildew and fire blight, showed a protein pattern comparable to that of the IWF from V. inaequalis infected leaves from cultivar Elstar indicating the constitutive production at least of some of the pathogenesis-related proteins in the resistant cultivar.     

Proteome analysis of Fusarium infection in emmer grains (Triticum dicoccum)

The fungal infection of emmer grain (Triticum dicoccum) with Fusarium graminearum and Fusarium culmorum was investigated at the level of the proteome. High‐resolution two‐dimensional gel electrophoresis and mass spectrometry were used to identify proteins that were differentially expressed in response to fungal infection of emmer. Moreover, the effects of natural field conditions at two locations on the carbon and nitrogen contents and the mycotoxin concentration of emmer grains were evaluated. Inoculation of emmer with a mixture of the two Fusarium species led to infection of the ears, with deoxynivalenol concentrations up to 10 mg kg^?1 in the grain. Carbon concentration and crude protein content were not significantly changed, but 10 distinct proteins changed in abundance. Stress‐related proteins, such as a serine protease inhibitor, a thaumatin‐like protein that reduced fungal growth and the starch hydrolysis β‐amylase increased upon infection, whereas the stress‐related proteins peroxidase, peroxiredoxin, a starch‐synthesis protein (a glycosyltransferase) and a fungal cell wall degrading protein (a chitinase) decreased. Furthermore, levels of three storage proteins in emmer grains were affected by Fusarium infection: α‐gliadin decreased and two globulins increased upon infection.     

Effect of phytoplasmal infection on photosystem II efficiency and thylakoid membrane protein changes in field grown apple (Malus pumila) leaves

We have studied the effect of the apple proliferation phytoplasmal infection on some features of the thylakoids from field grown apple (Malus pumila) leaves. Changes in photosynthetic pigments, soluble proteins, ribulose-1,5-bisphosphate carboxylase, nitrate reductase, photosynthetic activities and thylakoid membrane proteins were investigated. The level of total chlorophyll and carotenoids were reduced in phytoplasma-infected leaves. Similar results were also observed for soluble proteins and ribulose- 1,5-bisphosphate carboxylase activity. The in vivo nitrate reductase activity was significantly reduced in infected leaves. When various photosynthetic activities were followed in isolated thylakoids, phytoplasmal infection caused marked inhibition of whole chain and photosystem II activity while the inhibition of photosystem I activity was only marginal. The artificial exogenous electron donors, diphenyl carbazide and hydroxylamine significantly restored the loss of photosystem II activity in infected leaves. The same results were obtained when F_v/F_m was evaluated by chlorophyll fluorescence measurements. The marked loss of photosystem II activity in infected leaves could be due to the loss of 47, 33, 28–25, 23 and 17 kDa polypeptides. It is concluded that phytoplasmal infection inactivates the donor side of photosystem II. This conclusion was confirmed by immunological studies showing that the content of the 33 kDa protein of the water-splitting complex was diminished significantly in infected leaves.     

Phytoplasma [Stolbur-subgroup (Bois Noir-BN)] infection inhibits photosynthetic pigments, ribulose-1,5-bisphosphate carboxylase and photosynthetic activities in field grown grapevine (Vitis vinifera L. cv. Chardonnay) leaves

In this work we have studied the influence of phytoplasma-induced grapevine yellows (yellowing) on some features of the thylakoids from field grown grapevine (Vitis vinifera L. cv. Chardonnay) leaves. Changes in photosynthetic pigments, soluble proteins, ribulose-1,5-bisphosphate carboxylase, photosynthetic activities and thylakoid membrane proteins were investigated. The level of total chlorophyll and carotenoids, on a unit fresh weight basis, showed a progressive decrease in phytoplasma infected leaves. Similar results were also observed for total soluble proteins and ribulose-1,5-bisphosphate carboxylase activity. When various photosynthetic activities were followed in isolated thylakoids, phytoplasma infection caused marked inhibition of whole chain and photosystem (PS) II activity. Smaller inhibition of PSI activity was observed even in severely infected leaves. The artificial exogenous electron donors, DPC and NH₂OH significantly restored the PSII activity in both mild and severely infected leaves. The same results were obtained when F_v/ F_m was evaluated by Chl fluorescence measurements. The marked loss of PS II activity in infected leaves was evidently due to the loss of 33, 28–25, 23, 17 and 10 kDa polypeptides. This conclusion was confirmed by immunological studies showing that the content of the 33 kDa protein of the water-splitting complex was diminished significantly in infected leaves. Phytoplasma infection induced a fast degradation of LHCP II which became visible as yellowish colour in leaves.     

Expression of defence‐related genes in stems and leaves of resistant and susceptible field pea (Pisum sativum) during infection by Phoma koolunga

The differential expression of 13 defence‐related genes during Phoma koolunga infection of stems and leaves of susceptible versus resistant field pea (Pisum sativum) was determined using qRT‐PCR. Expression, in terms of relative mRNA level ratios, of genes encoding ferredoxin NADP oxidoreductase, 6a‐hydroxymaackiain methyltransferase (hmm6), chalcone synthase (PSCHS3) and ascorbate peroxidase in leaves and stems differed during 6–72 hours post‐inoculation (hpi) and reflected known host resistance levels in leaves versus stems. In comparison to the susceptible genotype, at 24, 48 and 72 hpi, two genes, hmm6 (122.43‐, 206.99‐ and 32.25‐fold, respectively) and PSCHS3 (175.00‐, 250.13‐ and 216.24‐fold, respectively), were strongly up‐regulated in leaves of the resistant genotype, highlighting that resistance against P. koolunga in field pea is governed by the early synthesis of pisatin. At 24 hpi, leaves infected by P. koolunga showed clear differences in expression of target genes. For example, the gene encoding a precursor of the defensin ‘disease resistance response protein 39’ was substantially down‐regulated in leaves of both the susceptible and the resistant genotypes inoculated with P. koolunga. This contrasts with other studies on another pea black spot pathogen, Didymella pinodes, where this same gene is strongly up‐regulated in leaves of resistant and susceptible genotypes. The current study provides the first understanding of defence‐related genes involved in the resistance against P. koolunga, opening novel avenues to engineer new field pea cultivars with improved leaf and stem black spot disease resistance as the basis for developing more effective and sustainable management strategies.     

Water uptake and proline index as indicators of predisposition in tomato plants to Phytophthora nicotianae infection as influenced by abiotic stresses

To identify quantitative indicators of predisposition and stress development in tomato (Lycopersicon esculentum Mill. cv. Counter) plants infected by Phytophthora nicotianae Breda de Haan, we examined plants grown under stress-inducing nutrient conditions exposed to different levels of radiation and infection. The plant's response was investigated by analysing plant growth, proline content and disease development. From all plant growth parameters investigated, only leaf surface area and total fresh mass showed a response to radiation and infection, and both indicators were strongly correlated. However, because of the destructive character of these indicators, non- or minimal destructive indicators were sought. To this end, here we report that water uptake per plant (WP) and water uptake per plant and day (WPD) and proline content of leaves represent useful tools to assess plant health status during growth.     

Spread of seed-borne <Emphasis Type="Italic">Erwinia rhapontici</Emphasis> in bean,pea and wheat

Studies were conducted in the laboratory and greenhouse to determine the distribution of Erwinia rhapontici in plants arising from naturally infected seeds of pea or artificially inoculated seeds of bean and wheat, and whether the pathogen is transmitted to the subsequent generation of seeds. Infected seeds were planted in pots of Cornell mix in the greenhouse, and sampled at specified intervals throughout the plant growth cycle (seedling stage, elongation stage, flowering stage, seed formation stage, and maturity). Plating of surface sterilized lateral roots, tap roots, basal stems, mid-stems, apical stems, petioles, pods, and seeds of pea and bean, and of lateral roots, sub-crown internodes, basal stems, mid-stems, apical stems, peduncles, glumes, and seeds of wheat revealed that the bacterial pathogen spread from infected seeds to the lower parts of the plant tissues, but failed to spread further to the seeds produced on these plants. The study concludes that E. rhapontici did not establish systemic infection throughout the plants. Possible mechanisms of infection of seeds are discussed.     

Proteomic analysis of mycelial proteins from <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

The soilborne pathogen Rosellinia necatrix causes white root rot, a serious disease of various trees, and is extremely difficult to control. In this study, using one-dimensional electrophoresis coupled with nanoliquid chromatography-electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF-MS/MS), we identified 696 proteins from R. necatrix mycelium (KACC 40445) grown in liquid culture. In addition, 573 proteins were assigned to at least one gene ontology term including 26 functional groups. Most were related to catalytic activity in the molecular function category. This proteomic data set advances understanding of R. necatrix biology and will inform further investigations to manage white root rot using novel strategies.     

瓠瓜果斑病症状及其病原菌的鉴定

为有效防控瓠瓜果斑病,自浙江省象山县田间采集具有典型果斑病症状的瓠瓜样本,对其进行病原菌分离、形态观察、致病性测定及分子生物学鉴定,并利用特异性引物PL1/PL2 PCR扩增和基质辅助激光解吸电离飞行时间质谱（matrix assisted laser desorption ionization time-of-flight massspectrometry,MALDI-TOF-MS）技术对其病原菌进行亚群鉴定。结果表明：瓠瓜果斑病田间典型症状是发病叶片和果实上病斑由水渍状小斑点逐渐发展为伴有黄色晕圈的褐色不规则病斑,果实腐烂、有臭味。通过菌体形态和培养特性观察、PCR鉴定、16S rDNA序列分析和7个看家基因的系统发育分析将其病原菌鉴定为西瓜噬酸菌Acidovorax citrulli。从瓠瓜上分离的菌株均属于西瓜噬酸菌亚群I,从西瓜上分离的菌株均属于西瓜噬酸菌亚群II。     

应用A-蛋白诱捕修饰和羊抗兔血清诱捕双修饰法研究马铃薯X病毒

A-蛋白和羊抗兔血清应用于免疫电镜,检测马铃薯X病毒(PVX)粗汁液,灵敏度比诱捕修饰法高;而且加羊抗兔血清后,病毒粒体比诱捕修饰法明显加粗,在电镜放大2500倍时,就能清晰地观察到。用诱捕双修饰法检测PVX,证实在寄主植物的叶、茎和根中均存在,以叶内含量最高。接种在普通烟上第三天,接种叶就能检测到PVX,接种15天后,寄主体内病毒能达到较高浓度,而且高浓度一直可保持2个月以上;PVX在不同寄主中的含量不同,以普通烟中病毒浓度最高,心叶烟、番茄和黄花烟中次之;昆诺阿藜、千日红和大椒中含毒量极抵。用诱捕双修饰法检测马铃薯薯块休眠芽中的病毒获得成功。     

Suitability of Ten Plant Baits for the Rapid Detection of Pathogenic Pythium Species in Hydroponic Crops

Ten types of plant baits were tested in the laboratory to assess their capacity to detect pathogenic Pythium species. These were orange tree leaves, tomato leaves, pepper leaves, geranium leaves, Bermuda grass leaves, pine needles, immature carnation petals, hemp-seed cotyledons, pepper and cucumber fruits. The Pythium spp. tested were P. aphanidermatum, P. irregulare and Pythium group F from hydroponic market garden crops in the Poniente region of Almería (south-east Spain). The test consisted of observing the velocity at which five baits were colonized and the day of colonization of the first bait. Results indicated that the slowest baits to be infected were immature carnation petals and pine needles. These two, together with Bermuda grass leaves, were also the baits infected in lowest number, such that practically no further infection was produced in the baits after the fifth day of contact with the inoculated water. The other plant baits tested were equally suitable for detection of Pythium spp. over the first two days, although only orange leaves and hemp-seed cotyledons were infected on the first day.     

Fungal derived cytokinins are necessary for normal Ustilago maydis infection of maize

Phytohormones derived from fungi play a key role in regulating plant–pathogen interactions; however, deciphering the separate contributions of pathogen and plant during infection has been difficult. Here, the Ustilago maydis–Zea mays pathosystem was used to investigate this chemical exchange. Ustilago maydis, the causative agent of maize smut, produces cytokinins (CK), which are a group of phytohormones responsible for directing plant development. The characteristic symptom of smut disease is the formation of tumours composed of plant and fungal tissue. Isopentenyltransferase (IPT) catalyses the rate‐limiting step in CK biosynthesis, and U. maydis strains in which the sole tRNA‐ipt gene was deleted no longer produced CKs. These deletion strains elicited fewer, smaller tumours than the pathogenic strain SG200. High performance liquid chromatography‐electrospray ionization tandem mass spectrometry (HPLC‐ESI MS/MS) was used to detect and quantify phytohormone levels in infected tissue. This revealed that key hormone changes in SG200 infections were not present in infections by deletion strains, suggesting that CK production by U. maydis is required for the altered phytohormone profile in infected tissue relative to uninfected tissue. Separate analyses indicated that U. maydis tRNA‐ipt mutants might be altered in their ability to metabolize CKs taken up from the environment. Mining the U. maydis genome identified genes encoding putative CK signalling and biosynthesis proteins.     

Screening for Differential Gene Expression in Atropa belladonna Leafy Gall Induced Following Rhodococcus fascians Infection

To better characterise at the molecular level the nature of plant responses to infection by Rhodococcus fascians PCR-based differential display patterns of Atropa belladonna leafy gall (LG) and non-infected plant tissues were compared. Six differentially expressed genes were identified and their altered expression was confirmed by RT-PCR. Three of them corresponded to up-regulated genes which encode proteins involved in plant defence. The three remaining cDNA fragments which correspond to down-regulated genes in LG, encoded proteins with similarity to a multicystatin, a miraculin and a methallothionein-like protein, respectively. Upon elimination of the bacteria from infected plant tissue, the expression of up-regulated genes was maintained, whereas expression of down-regulated genes resumed suggesting a potential role of these up-regulated genes in plant growth and development.     

Biochemical and physiological responses to long‐term Citrus tristeza virus infection in Mexican lime plants

Reactions that occur when a plant is subjected to Citrus tristeza virus (CTV) infection often result in triggering of numerous defence mechanisms to fight the infection. The reactions vary according to virus strain, host genotype, time of exposure to the infection and environmental conditions. To date, no study has examined in detail the consequences of 10‐year exposure to CTV infection on the biochemical and physiological status of susceptible Mexican lime plants (Citrus aurantifolia). To understand the reaction of such plants, changes in nutrient status, total proteins, enzyme activity involved in scavenging of reactive oxygen species, photosynthetic and transpiration rates, chlorophyll content, membrane permeability and water content were analysed in plants infected with different CTV isolates and in healthy plants. The activity of superoxide dismutase and polyphenol oxidase significantly decreased in the infected leaves, and membrane permeability was lower in the infected plants. Macro‐ and micronutrient elements were significantly changed: concentrations of leaf nitrogen, zinc, magnesium and iron were elevated but potassium concentration depressed in comparison to noninfected control leaves. Levels of other analysed nutrient elements, enzymes, photosynthesis and stomatal conductance, chlorophyll content and relative water content were unchanged. Clear physiological changes were found among infected and noninfected control plants but none between plants infected with different CTV isolates. The data suggest that some of the defence mechanisms investigated here were suppressed due to the continuous and long‐term pressure of biotic stress.     

Host–pathogen interaction between Phytophthora infestans and Solanum nigrum, S. villosum, and S. scabrum

Potato and tomato are the two major hosts for Phytophthora infestans causing late blight. The susceptibility of leaves and whole plants of Solanum nigrum, S. villosum, and S. scabrum to infection by P. infestans was tested under laboratory conditions. Out of 39 plants representing 38 different S. nigrum accessions, 16 were highly resistant (seven accessions did not show any symptoms of infection, nine were highly resistant showing necrotic lesions in the place of infection), and 23 plants of S. nigrum were colonized by, at least, 1 of the 2 isolates of P. infestans (17 accessions were infected with two P. infestans isolates, and 6 accessions showed different reactions depending on the isolate used for inoculation). Three accessions of S. villosum, and one accession of S. scabrum were tested and did not show any symptoms of infection. The majority of S. nigrum accessions infected by P. infestans in a detached leaf assay were also infected in the whole plant assay. The reaction of field- and greenhouse-grown plants to inoculation with P. infestans in detached leaf assays was similar, but in some cases leaves from field-grown plants reacted as resistant in comparison with the leaves from greenhouse-grown plants, which were susceptible.