双生病毒——一类值得重视的植物病毒

 双生病毒是世界范围内广泛发生的一类植物单链DNA病毒,已在多种作物上引起严重危害。重组是导致双生病毒变异和病害流行的重要原因。近年来,在单组份双生病毒中发现有新型卫星DNA分子——DNAβ,DNAβ是病毒致病所必需的。本文就双生病毒的危害性、病毒的重组、DNAβ的发现及与致病性的关系、双生病毒与伴随的小分子DNA形成病害复合体、双生病毒DNA-A和小分子DNA的进化关系等问题进行了简要评述。     

Expression of defence genes in stolbur phytoplasma infected tomatoes,and effect of defence stimulators on disease development

In tomato, the stolbur disease caused by ‘Candidatus Phytoplasma solani’ alters developmental processes resulting in malformations of both vegetative and reproductive organs, two stolbur phytoplasma strains PO and C induce mutually distinct symptoms. The aim of the present study was to determine the effect of stolbur phytoplasma-infection on the Salicylic (SA) and Jasmonic (JA) acids hormone signalling pathways and to assess whether pre-activation of these defence pathways could protect tomato against the stolbur disease development. Expression of SA- and JA-dependent marker genes was studied in tomato by qRT-PCR. Results indicated that the SA-mediated defence response was activated by the stolbur phytoplasma strains PO and C in contrast to the JA-dependent defence pathway which was repressed by strain PO but activated by strain C. The two stolbur strains, PO and C, generated different responses, suggesting that the two strains might have distinct virulence factors, in agreement with the fact that they induce distinctive symptoms. In stolbur PO-infected tomato, pre-activation of the JA-dependent defence pathway by methyl jasmonate (MeJA) before infection had no effect on the disease development whereas pre-activation of the SA-dependent defence pathway by treatment with benzothiadiazole (BTH) prior to graft-inoculation of the phytoplasma resulted in a minor delay in phytoplasma multiplication and symptom production. As grafting implicates a high inoculum as compared to insect inoculation, it would be of interest to test BTH treatment in natural conditions.     

Sulphur supply impairs spread of Verticillium dahliae in tomato

Vascular wilt caused by the soil-borne fungus Verticillium dahliae is a major yield and quality-limiting disease across a broad spectrum of crop plants worldwide. Sulphur-enhanced plant defence mechanisms provide an opportunity to effectively and environmentally safely constrain the wilt disease levels in planta. To evaluate the influence of sulphur nutrition on the protective potential of these mechanisms, two near-isogenic tomato genotypes differing in fungal susceptibility, were treated with low or supra-optimal sulphur supply. Microscopic analysis revealed a significant sulphur-induced decrease in the amount of infected vascular cells in both genotypes. However, plant shoot and severely pathogen-affected root growth did not display this distinct alleviating influence of sulphur nutrition. Rates of leaf photosynthesis were impeded by Verticillium dahliae infection in both genotypes especially under low sulphur nutrition. However, assimilate transport rates in the phloem sap were enhanced by fungal infection more in the resistant genotype and under high sulphur nutrition suggesting a stronger sink for assimilates in infected plant tissues possibly involved in sugar-induced defence. A SYBR Green-based absolute quantitative Real-Time assay using a species-specific primer was developed which sensitively reflected sulphur nutrition-dependent changes in fungal colonization patterns. High sulphur nutrition significantly reduced fungal spread in the stem in both tomato genotypes. Concentrations of selected sulphur-containing metabolites revealed an increase of the major anti-oxidative redox buffer glutathione under high sulphur nutrition in response to fungal colonization. Our study demonstrates the existence of sulphur nutrition-enhanced resistance of tomato against Verticillium dahliae mediated by sulphur-containing defence compounds.     

Effect of plant elicitors on the reproduction of the root-knot nematode Meloidogyne chitwoodi on susceptible hosts

Sustainable strategies for plant-parasitic nematode control are required to reduce dependence on chemical nematicides. Foliar application of various compounds can induce a systemic defence response that reduces nematode infestation. The effects of benzothiadiazole (BTH), β-aminobutyric acid (BABA), jasmonates (cis-jasmone and methyl jasmonate) and salicylic acid (SA) in the development and reproduction of the root-knot nematode Meloidogyne chitwoodi in tomato plants were assessed. The effects of BTH and of the jasmonates were further tested on potato plants. Pot assays were conducted using tomato plants cv. Tiny Tim or potato cv. Désirée treated with foliar sprays and inoculated with 300 second stage juveniles. Nematode development and reproduction were assessed 21 and 45 days after inoculation. Treatment with SA had a negative effect on nematode development in tomato plants but did not affect reproduction and methyl jasmonate treatment was the most effective in reducing nematode penetration (58 %). Nematode development was significantly affected in potato plants sprayed with cis-jasmone. Nematode penetration was reduced by 90, 67 and 81 % in plants treated with BTH, cis-jasmone and methyl jasmonate respectively, although the reproduction factor (Rf) was only significantly lower in the BTH treatment (Rf?=?7.6) when compared to the control (Rf?=?18.1). Our results suggest that both the SA and JA pathways play an important role in plant defence mechanisms against root-knot nematode development and reproduction for both plants, and should be considered in the design of integrated pest management approaches.     

Efficient, long-lasting resistance against the soft rot bacterium Pectobacterium carotovorum in calla lily provided by the plant activator methyl jasmonate

The potential of three externally applied chemical plant activators, Bion, BABA and methyl jasmonate, known to act only through the plant defence system and not on the pathogen directly, to induce resistance against wild-type Pectobacterium carotovorum was examined in white-flowered calla lily ( Zantedeschia aethiopica ). Following a 24-h induction period, plants were challenge-inoculated with P. carotovorum , originally isolated from calla lily or potato plants, previously transformed using a gfp broad-host-range promoter-probe vector. After another 24 h, Bion treatment (10  µ g mL⁻¹, as a drench) reduced disease symptoms more than sixfold and bacterial proliferation by four orders of magnitude. BABA treatment (5–10  µ g mL⁻¹, also as a drench) reduced the rate of infection by 75–85%. However, the protection afforded by both inducers did not persist. Also, at higher concentrations both displayed a phytotoxic effect. By contrast, methyl jasmonate (10 m m , applied as a leaf spray) completely inhibited P. carotovorum development in calla lily leaves and afforded a long-lasting effect. It is suggested that the defence response of calla lily against P. carotovorum involves the SA-signalling pathway in the short term, but the jasmonate/ethylene-signalling pathway is required for durable protection.     

The transmission of geminiviruses by Bemisia tabaci

The cosmopolitan whitefly species, Bemisia tabaci (Gennadius) and Trialeurodes taporariorum (Westwood) have always been regarded as pests to a large range of worldwide crops. Both species are capable of transmitting plant viruses, with T. vaporariorum being the vector of only a few ‘clostero’-like viruses and B. tabaci the vector of viruses in several groups. The largest group of viruses transmitted by B. tabaci are the geminiviruses and B. tabaci is known to transmit around 60 members. Until recently, B. tabaci had been associated with only a limited range of host plants within any one region, although its total potential host range was large. Virus transmission was confined within the plant host range of each regional population of B. tabaci. The emergence of the polyphagous ‘B’ biotype of B. tabaci with its increased host range of more than 600 plant species, has resulted in geminiviruses infecting previously unaffected crops. As the ‘B’ biotype spreads further into Europe, European field and glasshouse crops have been shown to be susceptible to whitefly-transmitted viruses already endemic to other parts of the world. More than 20 colonies of B. tabaci, including both ‘B’ and non-‘B’ biotypes from disparate global locations have been compared for their ability to transmit more than 20 geminiviruses. All but two highly host-specific colonies were capable of transmitting most geminiviruses tested. However, some viruses were transmitted more efficiently than others. The virus coat protein or capsid is essential for vector recognition and transmission. By comparing transmissible viruses at the molecular level to viruses that are no longer whitefly-transmissible, the active epitope on the virus coat protein could be identified for designing future virus control strategies.     

Soilborne viruses: advances in virus movement,virus induced gene silencing,and engineered resistance

Until recently soilborne plant viruses were considered important only because they are causative agents for agricultural diseases. In recent years, soilborne plant viruses have played a significant role in advancing research into mechanisms of plasmodesmata transport, gene silencing, and engineered resistance to plant pathogens. Three different mechanisms by which viruses move through plasmodesmata have been identified using dianthoviruses, nepoviruses, and benyviruses. The infectious clone of the tobravirus Tobacco rattle virus (TRV) has become an important tool for studying virus induced gene silencing and may be a tool to silence meristematic gene expression. Investigations of soilborne viruses may enable the development of new strategies for control of soilborne diseases. The potential use of pathogen-derived resistance to control soilborne viruses is currently being explored in several laboratories.     

A geminivirus causing vein yellowing of Ageratum conyzoides in Singapore

A vein-yellowing disease of Ageratum conyzoides in Singapore was shown to be caused by a geminivirus, here named ageratum yellow vein virus (AYVV), which was transmitted by the whitefly Bemisia tabaci but not by inoculation with sap or through seed. AYVV particles (30 × 20 nm) are serologically related to those of other whitefly-transmitted geminiviruses, and reacted with some monoclonal antibodies elicited by particles of African cassava mosaic or Indian cassava mosaic geminiviruses. However, the epitope profile of AYVV differed from the profiles of these viruses, and from those of geminiviruses from vein yellowing-affected A. conyzoides from India and from yellow leaf curl-affected tomato from either Singapore or India. The results provide further evidence of antigenic differences among geminiviruses that cause similar diseases in the same plant species in different geographical regions.     

The role of jasmonic acid signalling in wheat (Triticum aestivum L.) powdery mildew resistance reaction

Jasmonic acid (JA) signalling plays an important role in plant resistance to pathogens. Previously, JA has been found to play a role in induced disease resistance to necrotrophic pathogens in various plant species, but current researches showed that JA also enhanced resistance to biotrophic pathogens. However, its role in wheat (Triticum aestivum L.) powdery mildew (Blumeria graminis f. sp. tritici, Bgt) resistance reaction is largely unknown. To settle this issue, several typical powdery mildew resistant and susceptible wheat varieties were employed. The sensitivity to exogenous methyl jasmonate (MeJA) to wheat powdery mildew resistance, the concentration fluctuation of endogenous JAs after Bgt inoculation, and the expression profiles of nine pathogenesis-related protein genes (PR genes) after MeJA and Bgt treatments were studied systematically. Exogenous MeJA significantly enhanced the powdery mildew resistance of the susceptible varieties. After inoculation with Bgt, endogenous JAs accumulated rapidly, reached the maxima at 2 to 5 h post-inoculation (hpi), then decreased rapidly, and the concentration was almost the same as that of un-inoculated control at 96 hpi. The expression levels of the nine PRs were measured by real time quantitative RT-PCR (qRT-PCR) at different time points after MeJA application or Bgt inoculation respectively. The MeJA and Bgt strongly activated PR1, PR2, PR3, PR4, PR5, PR9, PR10 and Ta-JA2, but almost didn’t affect Ta-GLP2a. The induced powdery mildew resistance was positively correlated with the activated PR genes. JA plays a positive role in defence against Bgt. JA is a signalling molecule in wheat powdery mildew resistance and future manipulation of this pathway may improve powdery mildew resistance in wheat breeding.     

Involvement of kaempferol in the defence response of virus infected Arabidopsis thaliana

The roles of several phenolic compounds in plant defence response have been extensively studied, yet little is known about the role of flavonoids in plant-virus interaction. Quantitative and qualitative changes of selected phenolics in Arabidopsis thaliana induced by Cucumber mosaic virus containing satellite RNA (CMVsat) infection were analysed accompanied by plant hormone, chalcone synthase and pathogenesis-related gene expression analysis. Lower leaves of infected plants had a lower concentration of total phenolics compared to control plants. The concentration of kaempferol in upper leaves of all infected plants was significantly lower compared to control plants, while the expression of the chalcone synthase gene in those leaves was in most cases upregulated. All infected plants had a higher concentration of indole-3-acetic acid in lower leaves, which was accompanied with a lower concentration of kaempferol in upper leaves. Our research demonstrates a correlation between kaempferol and indole-3-acetic acid in response to CMVsat infection in Arabidopsis. We demonstrated two different metabolic patterns in infected plants suggesting the activation of two different defence responses. We also propose kaempferol to be an important part of the auxin-dependent defence response which limits systemic movement of CMVsat and that this defence response is activated prior to the well-known salicylic acid dependent defence response. Further research on kaempferol and its role in Arabidopsis-CMVsat interaction will improve our understanding on the role of flavonoids in plant defence.     

Ethylene signalling is essential for the resistance of Nicotiana attenuata against Alternaria alternata and phytoalexin scopoletin biosynthesis

The phytohormone ethylene plays an important role in plant defence responses to pathogen attack. When infected by the necrotrophic fungal pathogen Alternaria alternata (tobacco pathotype), which causes severe diseases in Nicotiana species, the wild tobacco plant Nicotiana attenuata accumulates a high amount of the jasmonate (JA)‐dependent phytoalexin scopoletin to defend itself against this fungal pathogen. However, it is still not known whether ethylene signalling is also involved in scopoletin biosynthesis and the resistance of N. attenuata. After infection, ethylene biosynthetic genes were highly elicited. Furthermore, plants strongly impaired in ethylene biosynthesis or perception had dramatically decreased scopoletin levels, and these plants became more susceptible to the fungus, while A. alternata‐elicited JA levels were increased, indicating that the decreased defence responses were not due to lower JA levels. Thus, it is concluded that after infection, ethylene signalling is activated together with JA signalling in N. attenuata plants and this subsequently regulates scopoletin biosynthesis and plant resistance.     

高通量测序技术在鉴定木本植物双生病毒中的应用

高通量测序技术(next-generation sequencing,NGS)平台提供了一种高效、快速、低成本、深度测序DNA的解决方案,2009年该技术开始被应用于植物病毒学领域,包括新病毒的发现,病害病原的鉴定,病毒基因组多样性及进化的研究,显著加快了植物病毒学的发展进程。迄今,应用高通量测序技术已经成功鉴定了上百种新的植物病毒和类病毒。双生病毒是一类对多种作物造成毁灭性危害的DNA病毒,多发生于草本作物。然而利用高通量测序技术,从柑橘、葡萄、苹果和桑树等多种多年生木本植物中检测到了新的双生病毒,显示出了高通量测序技术所独有的、传统检测技术所不具备的优势。本文围绕高通量测度技术在植物病毒学领域的应用进行概述,重点阐述NGS用于检测木本植物双生病毒的几个实例。     

Phenolic compounds as defence response of pepper fruits to Colletotrichum coccodes

Qualitative and quantitative changes of individual and total phenolics induced by Colletotrichum coccodes fungal infection have been studied in two susceptible sweet pepper cultivars ‘Soroksari’ and ‘Bagoly’, and the role of soluble phenolic compounds in plant's defence mechanism has been evaluated. Three distinct parts were analysed on pepper fruit: healthy tissue, anthracnose lesion, and bordering tissue, and individual phenolic compounds have been identified with the use of HPLC-MS system. In pepper fruit pericarp 21 phenolic compounds have been determined; the prevalent apigenin, quercetin and luteolin glycosides, chlorogenic acid and one chrysoeriol glucoside. C. coccodes infection increased the accumulation of chlorogenic acid, chrysoeriol glucoside, quercetin and luteolin glycosides in infected bordering tissue of both analysed pepper cultivars compared to healthy pepper tissue or symptomatic spot. Total apigenin derivatives did not show a significant increase in bordering tissue compared to the healthy pepper fruit in contrast to other groups of phenolics. This suggests a lesser role of apigenin glycosides in pepper plant defence against the Colletotrichum fungus. Intense phenolic synthesis was characteristic for the bordering zone between the healthy and infected plant tissue resulting in higher total phenolic content which might hinder the fungus to spread from the infected cells into the healthy tissue.     

Inhibition on brown rot disease and induction of defence response in harvested peach fruit by nitric oxide solution

Nitric oxide (NO) is an important signal molecule involved in numerous plant responses to biotic and abiotic stresses. The effect of nitric oxide (NO) solution on pathogen infection and defence response of peach (Prunus persica (L.) Batsch) fruit against brown rot disease caused by Monilinia fructicola was investigated. The results showed that 15 μmol l^?1 NO solution did not significantly inhibit spore germination, germ tube length or pathogenicity of M. fructicola, but significantly reduced disease incidence and lesion areas in the fruit. Although 100 μmol l^?1 NO solution effectively inhibited the spore germination, germ tube elongation and pathogenicity of M. fructicola, the high concentration of NO solution caused damage to the fruit. Moreover, 15 μmol l^?1 NO enhanced the activities of chitinase (CHI) and β-1,3-glucanase (GNS) in the fruit. RT-PCR analysis showed that the expression of four genes, CHI, GNS, pathogenesis-related protein 1 and 10 genes (PR-1, PR-10) all increased after NO treatment. Conversely, pretreatment with 100 μmol l^?1 NO scavenger, 2-4-carboxyphenyl-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), rendered the fruit relatively susceptible to pathogen infection and inhibited the defence response of the fruit. These results suggest that NO solution treatment can protect peach fruit from pathogen infection by inducing the activities of the defence enzymes and the expression of PR genes.     

Identification by suppression subtractive hybridization and expression analysis of Arabidopsis thaliana putative defence genes during Orobanche ramosa infection

A suppression subtractive hybridization strategy was used to identify genes that were induced 2 h after infection of Arabidopsis thaliana by broomrape (Orobanche ramosa) seedlings. Among these genes, the expression of twelve was analysed from the first hour to the seventh day of this compatible interaction by cDNA blot analyses. The twelve genes showed a transient expression, which occurred in seven cases as early as the first or second hour of interaction and ended 2 or 3 h later. Most of the proteins encoded by these genes are already known to be involved in different A. thaliana response pathways to pathogen attack. The first two genes to be induced (Rc kinase and ACaM5) encoded a receptor and a calmodulin, and could be involved in signal transduction. The two following genes encoding a sucrose carrier (SUC1) and a protein with a pectin methylesterase inhibitor domain were found to be overexpressed; their roles are consistent with plant defence emergence. The gst1, gst11 and peptide methionine sulfoxide reductase genes, which were turned on early, are known to play a role in the detoxification of reactive oxygen species. The accumulation of mRNAs for lox1, a latex allergen and a myrosinase binding protein could be related to a jasmonate dependent pathway, while genes for a class III peroxidase and a caffeoyl-CoA 3-O-methyltransferase, both likely to be involved in cell wall reinforcement, were also upregulated.     

Role of methyl jasmonate in the expression of mycorrhizal induced resistance against Fusarium oxysporum in tomato plants

Arbuscular mycorrhiza (AM) colonization led to a decrease in the severity of fusarium wilt disease caused by Fusarium oxysporum f. sp. lycopersici in tomato plants. The involvement of two plant defense hormones, namely methyl jasmonate (MeJA) and salicylic acid (SA), in the expression of mycorrhiza induced resistance (MIR) against this vascular pathogen was studied in the AM colonized and non-colonized (controls) plants. Activity of lipoxygenase (LOX), which plays a role in jasmonic acid (JA) biosynthesis, as well as levels of methyl jasmonate (MeJA) increased in AM colonized plants as compared to controls, but did not show any further changes in response to F. oxysporum inoculation. On the other hand, activity of phenylalanine ammonia lyase (PAL), which is an enzyme from salicylic acid (SA) biosynthetic pathway, as well as SA levels, increased in both controls and AM colonized plants in response to application of F. oxysporum spores. Hence the JA and not the SA signalling pathway appeared to play a role in the expression of MIR against this vascular pathogen. The resistance observed in AM colonized plants was completely compromised when plants were treated with the JA biosynthesis inhibitor salicylhydroxamic acid (SHAM). This confirmed that the AM-induced increase in JA levels was involved in the expression of resistance toward F. oxysporum. The SA response gene pathogenesis-related 1 (PR1) showed an increased expression in response to F. oxysporum infection in SHAM treated AM colonized plants as compared to plants that were not treated with this JA inhibitor. This suggested the possibility that JA inhibited SA responses, at least in the roots. AM colonization therefore appeared to prime plants for improved tolerance against the vascular pathogen F. oxysporum, which was mediated through the JA signalling pathway.