Development of a Pseudomonas syringae–Eutrema salsugineum pathosystem to investigate disease resistance in a stress tolerant extremophile model plant

To improve the ability to understand how plants respond to multiple and/or concurrent stresses, disease resistance was investigated in Eutrema salsugineum, an extremophile model plant that is highly tolerant of abiotic stress. Compared to Arabidopsis (Col‐0), both Yukon and Shandong Eutrema accessions exhibit increased resistance to Pseudomonas syringae pv. tomato DC3000 (Pst) and pv. maculicola (Psm), with Shandong Eutrema exhibiting greater resistance to Pst than Yukon Eutrema. RT‐PCR of the EsPR1 (Pathogenesis‐related 1) defence marker gene confirmed RNA‐Seq data that healthy Shandong Eutrema constitutively expresses EsPR1. The data suggests that Shandong Eutrema exists in a highly primed state of defence preparedness, as it displays heightened resistance compared to defence‐primed natural accessions of Arabidopsis (Can‐0, Bur‐0). Pathogen‐triggered PR1 expression was delayed in Yukon Eutrema; however, these plants were resistant to Pst suggesting that Yukon Eutrema employs a PR1‐independent mechanism to resist Pst. This study demonstrates that Eutrema is an excellent model to investigate biotic stress tolerance. The Eutrema–P. syringae pathosystem will facilitate future studies to understand how this extremophile tolerates both abiotic and biotic stress, and will allow exploration of the interplay of these responses to inform efforts to improve stress tolerance in crops.     

木寡糖提高拟南芥抗病性

木寡糖是一种植物源寡糖,对动物体的生理功能多且显著,而其是否作为寡糖素诱发植物防卫尚不明确.本文分析了不同浓度木寡糖诱导拟南芥植株对不同营养型病原的抗性.结果表明,木寡糖能诱发拟南芥对半活体病原细菌丁香假单胞菌番茄致病变种、活体病原烟草花叶病毒和死体病原真菌核盘菌的抗性,且具有剂量依赖效应,随着木寡糖浓度的提高到100...     

番茄细菌性斑点病菌无毒基因研究进展

番茄细菌性斑点病是影响番茄产量和品质的重要病害,Pseudomonas syringaepv.tomato(Pst)为其病原菌,其与番茄的互作系统是研究植物抗感病机理的典型模式系统。Pst存在2种无毒基因:avrPto和avrPtoB,它们编码的蛋白质均能与番茄抗性基因Pto编码的Ser-Thr蛋白激酶互作,符合Flor"基因对基因"学说。AvrPto和AvrPtoB在表达Pto的抗性植物中,与Pto互作,表现无毒功能,引发植物防御反应;而在缺失Pto的感病植物中,它们具有毒性,促进细菌的生长。本文综述了番茄细菌性斑点病菌无毒基因avrPto及avrPtoB的结构特点及其功能,这有助于了解病原物与植物的互作机制,对认识植物的感病性、抗病性以及植物防御反应都具有重要意义。     

Signal Interactions in Induced Resistance to Pathogens and Insect Herbivores

Plants are often simultaneously challenged by pathogens and insects capable of triggering an array of responses that may be beneficial or detrimental to the plant. The efficacy of resistance mechanisms can be strongly influenced by the mix of signals generated by biotic stress as well as abiotic stress such as drought, nutrient limitation or high soil salinity. An understanding of their biochemical nature, and knowledge of the specificity and compatibility of the signaling systems that regulate the expression of inducible responses could optimize the utilization of these responses in crop protection. Signaling conflicts and synergies occur during a plant's response to pathogens and insect herbivores, and much of the research on defense signaling has focused on salicylate- and jasmonate-mediated responses. We will review our results using tomato (Lycopersicon esculentum) in greenhouse and field studies that illustrate a trade-off between salicylate- and jasmonate-mediated signaling, and discuss research on strategies to minimize the trade-off that can occur following the application of chemical elicitors of resistance. In addition, there is evidence of another signaling system that mediates endogenous levels of ceramide in the plant. This signal is associated with programmed cell death and protection of tomato against the fungal pathogen Alternaria alternata f. sp. lycopersici.     

鞭毛素蛋白FliCxcv对水稻免疫反应的诱导功能鉴定

 为揭示来源于番茄细菌性斑点病菌(Xanthomonas campestris pv. vesicatoria, Xcv)菌株XV18鞭毛素(FliCxcv)作为一种病原相关分子模式(PAMP)诱导水稻免疫反应的功能,本研究对FliCxcv编码基因flicxcv进行了基因克隆、序列分析、原核表达、蛋白纯化和诱导活性测定。结果表明,通过PCR特异性扩增,从Xcv菌株XV18中克隆了1 200 bp的fliCxcv基因片段,其序列与GenBank中己测序菌株的完全一致。在大肠杆菌中对该基因全长、N端和C端截短序列进行了原核表达,并获得了纯化的FliCxcv全长及其截短蛋白。将纯化蛋白浸润接种到水稻品种日本晴叶片组织,发现FliCxcv全长及其截短蛋白均能诱导水稻叶片细胞死亡、H₂O₂产生以及防卫基因(OsPAL和OsPR1b)表达等免疫反应,但诱导活性存在差异。因此,本研究验证了FliCxcv具有激发水稻细胞免疫反应的PAMP功能,为水稻免疫诱导制剂的研发提供了材料。     

Reduction of Bacterial Speck (Pseudomonas syringae pv. tomato) of Tomato by Combined Treatments of Plant Growth-promoting Bacterium, Azospirillum brasilense, Streptomycin Sulfate, and Chemo-thermal Seed Treatment

Inoculation of tomato seeds with the plant growth-promoting bacterium Azospirillum brasilense, or spraying tomato foliage with A. brasilense, streptomycin sulfate, or commercial copper bactericides, separately, before or after inoculation with Pseudomonas syringae pv. tomato, the casual agent of bacterial speck of tomato, had no lasting effect on disease severity or on plant height and dry weight. Seed inoculation with A. brasilense combined with a single streptomycin foliar treatment and two foliar bactericide applications at 5-day intervals (a third or less of the recommended commercial dose) reduced disease severity in tomato seedlings by over 90% after 4 weeks, and significantly slowed disease development under mist conditions. A. brasilense did not induce significant systemic resistance against the pathogen although the level of salicylic acid increased in inoculated plants. Treatment of tomato seeds that were artificially inoculated with P. syringae pv. tomato, with a combination of mild chemo-thermal treatment, A. brasilense seed inoculation, and later, a single foliar application of a copper bactericide, nearly eliminated bacterial leaf speck even when the plants were grown under mist for 6 weeks. This study shows that a combination of otherwise ineffective disease management tactics, when applied in concert, can reduce bacterial speck intensity in tomatoes under mist conditions.     

The tomato Pto gene confers resistance to Pseudomonas floridensis,an emergent plant pathogen with just nine type III effectors

A newly discovered bacterial species, Pseudomonas floridensis, has emerged as a pathogen of tomato in Florida. This study compares the virulence and other attributes of P. floridensis to Pseudomonas syringae pv. tomato, which causes bacterial speck disease of tomato. Pseudomonas floridensis reached lower population levels in leaves of tomato as compared to the P. syringae pv. tomato strains DC3000 and NYT1. Analysis of the genome sequence of the P. floridensis type strain GEV388 revealed that it has just nine type III effectors including AvrPtoB_GEV388, which is 66% identical to AvrPtoB in DC3000. Five of these effectors have been previously reported to be members of a ‘minimal effector repertoire’ required for full DC3000 virulence on Nicotiana benthamiana; however, GEV388 grew poorly on leaves of this plant species compared to the DC3000 minimal effector strain. The tomato Pto gene recognizes AvrPtoB in race 0 P. syringae pv. tomato strains, thereby conferring resistance to bacterial speck disease. Pto was also found to confer resistance to P. floridensis, indicating this gene will be useful in the protection of tomato against this newly emerged pathogen.     

Involvement of jasmonic acid signalling in bacterial wilt disease resistance induced by biocontrol agent Pythium oligandrum in tomato

When the biocontrol agent Pythium oligandrum (PO) colonizes the rhizosphere, it suppresses bacterial wilt disease in tomato (Solanum lycopersicum cv. Micro‐Tom) caused by Ralstonia solanacearum, and a homogenate of its mycelia exhibits elicitor activity, inducing an ethylene (ET)‐dependent defence response in Micro‐Tom. Since salicylic acid (SA) and jasmonic acid (JA) play an important role in plant defence responses to pathogens, the involvement of SA‐ and JA‐dependent signal transduction pathways in resistance to R. solanacearum was investigated in tomato roots treated with a mycelial homogenate of PO. Bacterial wilt disease was also suppressed in tomato cv. Moneymaker treated with the PO homogenate. However, the SA‐inducible PR‐1(P6) gene was not up‐regulated in either Micro‐Tom or Moneymaker. SA did not accumulate in homogenate‐treated roots in comparison with distilled water‐treated controls, even 24 h after inoculation. Induced resistance against R. solanacearum was not compromised in SA‐non‐accumulating NahG transgenic plants treated with the PO homogenate. On the other hand, the expression of the JA‐responsive gene for the basic PR‐6 protein was induced in both tomato cultivars treated with the PO homogenate. Furthermore, quantitative disease assays showed that the induced resistance against R. solanacearum was compromized in PO homogenate‐treated jai1‐1 mutant plants defective in JA signalling. These results indicated that the JA‐dependent signalling pathway is required for PO‐induced resistance against R. solanacearum in tomato.     

Effects of para-aminobenzoic acid on bacterial speck symptom development and Pseudomonas syringae pv. tomato populations in tomato leaves

Para-aminobenzoic (PABA) is reported to induce resistance against a range of plant pathogens in different crops in a salicylic acid-dependent manner. However, factors affecting its efficacy are not well understood. Foliar PABA applications on tomato seedlings reduced lesion incidence caused by Pseudomonas syringae pv. tomato (Pst) in a dose-dependent manner in distal leaves up to 18 mM under controlled environment conditions, but only three out of six commercial processing tomato cultivars tested showed a response to PABA. Leaves in direct contact with 9 and 18 mM PABA of both PABA-responsive and PABA-nonresponsive cultivars showed phytotoxicity. In a PABA-responsive cultivar, one, two and three PABA applications were equally effective at reducing lesion incidence in distal leaves, but the duration of control only persisted for approximately 7 days. Although PABA application reduced lesion incidence in distal leaves, the Pst population in leaves was unaffected. Lesions on PABA-treated plants were larger than nontreated plants, and thus the proportion of leaf surface area with lesions was unaffected by PABA treatment. In in vitro assays, 18 and 72 mM PABA produced zones of inhibition against Pst 15 and 50% larger than the ethanol control, demonstrating direct antimicrobial effects of PABA. PABA application did not affect symptom development in a mixed infection of Pst or Xanthomonas spp. in one field experiment with a PABA-responsive cultivar. Further research is needed to understand why PABA was unsuccessful in the field before it is to be used as a practical disease management tool for foliar bacterial diseases of tomato.

     

Catalog of Micro-Tom tomato responses to common fungal, bacterial, and viral pathogens

Lycopersicon esculentum cultivar Micro-Tom is a miniature tomato with many advantages for studies of the molecular biology and physiology of plants. To evaluate the suitability of Micro-Tom as a host plant for the study of pathogenesis, Micro-Tom plants were inoculated with 16 well-known fungal, bacterial, and viral pathogens of tomato. Athelia rolfsii, Botryotinia fuckeliana, Oidium sp., Phytophthora infestans, and Sclerotinia sclerotiorum caused typical symptoms and sporulated abundantly on Micro-Tom. Micro-Tom was resistant to Alternaria alternata, Corynespora cassiicola, and Fusarium oxysporum. When Micro-Tom was inoculated with 17 isolates of Ralstonia solanacearum, many isolates induced wilt symptoms. Agrobacterium tumefaciens also was pathogenic, causing crown galls on stem tissue after needle prick inoculation. In Micro-Tom sprayed with Pseudomonas syringae pv. tomato, P. s. pv. tabaci, or P. s. pv. glycinea, bacterial populations did not increase, and yellow lesions appeared only on leaves sprayed with P. s. pv. tomato. Tomato mosaic virus, Tomato aspermy virus, and Cucumber mosaic virus systemically infected Micro-Tom, which developed symptoms characteristic of other cultivars of tomato after infection with the respective virus. These results indicated that Micro-Tom was generally susceptible to most of the important tomato pathogens and developed typical symptoms, whereas certain pathogens were restricted by either hypersensitive resistance or nonhost resistance on Micro-Tom. Therefore, an assortment of Micro-Tom–pathogen systems should provide excellent models for studying the mechanism of susceptible and resistant interactions between plants and pathogens.     

Magnesium oxide nanoparticles induce systemic resistance in tomato against bacterial wilt disease

Bacterial wilt is a serious problem affecting many important food crops. Recent studies have indicated that treatment with biotic or abiotic stress factors may increase the resistance of plants to bacterial infection. This study investigated the effects of magnesium oxide nanoparticles (MgO NP) on disease resistance in tomato plants against Ralstonia solanacearum, as well as its antibacterial activity. The roots of tomato seedlings were inoculated with R. solanacearum and then immediately treated with MgO NP; the treated plants showed very little inhibition of bacterial wilt. In contrast, when roots were drenched with a MgO NP suspension prior to inoculation with the pathogen, the incidence of disease was significantly reduced. Rapid generation of reactive oxygen species such as O₂^?˙ radicals was observed in tomato roots treated with MgO NP. Further O₂^?˙ was rapidly generated when tomato plant extracts or polyphenols were added to the MgO NP suspension, suggesting that the generation of O₂^?˙ in tomato roots might be due to a reaction between MgO NP and polyphenols present in the roots. Salicylic acid‐inducible PR1, jasmonic acid‐inducible LoxA, ethylene‐inducible Osm, and systemic resistance‐related GluA were up‐regulated in both the roots and hypocotyls of tomato plants after treatment of the plant roots with MgO NP. Histochemical analyses showed that β‐1,3‐glucanase and tyloses accumulated in the xylem and apoplast of pith tissues of the hypocotyls after MgO NP treatment. These results indicate that MgO NP induces systemic resistance in tomato plants against R. solanacearum.     

African wheat germplasm – a valuable resource for resistance to rust diseases

Known and unknown genes conferring seedling and adult plant resistance (APR) to leaf rust, stem rust and stripe rust were detected either singly or in combination in a set of 136 African wheat genotypes using multi-pathotype tests with characterized Australian Puccinia triticina (Pt), P. graminis f. sp. tritici (Pgt) and P. striiformis f. sp. tritici (Pst) pathotypes. Lines Beladi 132, IYN 68/9.44, Kenya Kifaru and Kenya Mbweha were postulated to carry resistance against multiple pathotypes of Pt, Pgt and Pst, whereas IAR/W/163-3, Grano Di Moggio Tipo 44 and Trigo 48 had resistance against all pathotypes tested in the current study. Field evaluation with the three rust pathogens detected low to high APR in more than 50% of lines, and while most tested positive with markers linked to known APR genes (csLV34, csLV46G22, TM10KASPAR, csGS, Cfb5006 and csSr2), many carried unidentified and useful resistance to all three rusts. Genetic analysis of F₃ mapping populations based on seven genotypes showed either monogenic or digenic inheritance of APR to leaf rust, stem rust and stripe rust. The lines postulated to carry effective uncharacterized seedling genes and APR genes are of great potential value in diversifying resistance to help achieve durable control of all three rust diseases of wheat.     

Regulation of defense-related enzymes associated with bacterial spot resistance in Tomato

Twenty tomato (Solanum lycopersicon) cultivars were screened for resistance against bacterial spot disease incited byXanthomonas axonopodis pv.vesicatoria under field conditions with and without pathogen infection. Screening was done by artificially inoculating aX. axonopodis pv.vesicatoria suspension to 4-week-old tomato seedlings and observing them for typical symptoms of the disease. Seedlings were categorized into highly resistant, resistant, susceptible and highly susceptible cultivars on the basis of disease incidence. Tomato cultivars were screened for defense-related enzymes, total phenols and lignin contents. The temporal patterns of all these enzymes were estimated with a moderately susceptible tomato cultivar. Native PAGE analysis of both peroxidase (POX) and polyphenol oxidase (PPO) was carried out for the time course of enzyme activities and also by selecting three different tomato cultivars, following infection with the pathogen. Based on the inducible amounts of these enzymes upon pathogen infection, the tomato cultivars were correlated with the disease incidence under field conditions. A significant (P≤0.05) correlation was observed between the degree of host resistance and the enzyme levels. In highly resistant tomato cultivars the enzyme levels, total phenols and lignin contents were increased in comparison with highly susceptible tomato cultivars. Isoform analysis of POX and PPO enzymes indicated a clear difference between resistant and susceptible tomato cultivars in the number of isoforms and also in the intensity of each isoform in the presence of pathogen infection. The possible regulation of defense-related enzymes in imparting host resistance is discussed. http://www.phytoparasitica.org posting March 11, 2008.     

Ectopic expression of barley constitutively activated ROPs supports susceptibility to powdery mildew and bacterial wildfire in tobacco

ROPs (also called RACs) are RHO-like monomeric G-proteins of plants, well-known as molecular switches in plant signal transduction processes, which are involved in plant development and a variety of biotic and abiotic stress responses. The barley (Hordeum vulgare) ROPs RACB, RAC1 and RAC3 have been shown to be involved in cellular growth, polarity and in susceptibility to the biotrophic barley powdery mildew fungus Blumeria graminis f.sp. hordei. We produced transgenic tobacco (Nicotiana tabacum) plants expressing constitutively activated (CA) mutants of the barley ROPs RACB and RAC3 to monitor the impact of heterologous ROP expression on cell polarity and disease susceptibility of tobacco. CA HvROPs influenced leaf texture, disturbed root hair polarity and induced cell expansion in tobacco. Both barley ROPs induced super-susceptibility to the compatible powdery mildew fungus Golovinomyces cichoracearum but only CA HvRAC3 induced super-susceptibility to the bacterial leaf pathogen Pseudomonas syringae pv. tabaci. Data suggest involvements of ROPs in tobacco cell expansion, polar growth and in response to bacterial and fungal leaf pathogens.     

球孢白僵菌定殖提高番茄对灰霉病抗性及其作用机理

为明确球孢白僵菌Beauveria bassiana定殖对番茄植株抗病性的影响及作用机理,采用灌根法将球孢白僵菌芽生孢子悬浮液接种于番茄植株内,并通过人工接种灰霉菌Botrytis cinerea评价球孢白僵菌定殖后番茄对灰霉病的抗性水平;检测灰霉菌胁迫下番茄植株不同位置叶片内球孢白僵菌的相对含量;测定番茄叶片内草酸氧化酶（oxalate oxidase,OXO）、几丁质酶（chitinase,CHI）和ATP合成酶（ATP synthase,atpA）3种抗病相关基因的表达量。结果表明,球孢白僵菌定殖能够提高番茄植株对灰霉病的抗性,接种灰霉菌第5天,番茄植株发病率、病斑直径和病情指数分别下降了61.6%、41.4%和26.4%;在灰霉菌胁迫下番茄植株内球孢白僵菌偏好于在病原菌感染位置定向聚集,并且引起植物抗病基因OXO、CHI和atpA的表达量上调。表明球孢白僵菌能通过内生定殖与植物互作提高植物抗病性,在植物病害生物防治领域有较大应用潜力。     

Specificity of polycllonal and monoclonal antibodies for the identification of Xanthomonas campestris pv. campestris

Polyclonal and monoclonal antibodies (PCAs and MCAs), produced to whole cells and flagellar extracts ofXanthomonas campestris pv.campestris (Xcc), respectively, were tested for specificity. In immunofluorescence microscopy (IF) the three PCAs tested, reacted at low dilutions with all Xcc strains, some other xanthomonads and non-xanthomonads. At higher dilutions most cross-reactivity with non-xanthomonad strains disappeared. However, the cross-reactivity with strains ofX. c. pv.vesicatoria (Xcv),X. c. pv.amoraciae (Xca) andX. c. pv.phaseoli var. fuscans (Xcpf) remained.Six MCA-producing cell clones viz. 20H6, 2F4, 18G12, 10C5, 17C12 and 16B5 were selected for specificity tests with an enzyme immunoassay (EIA), IF and a dot-blot immunoassay (DBI). None of the MCAs reacted with all Xcc strains in IF and EIA. In DBI, only MCAs 17C12 and 16B5 reacted with all Xcc strains. All six MCAs tested, cross-reacted in one of either tests with other pathovars ofX. campestris, such as Xcv or Xca. The MCAs were also tested in immunoblotting experiments using total bacterial extracts, cell envelope and flagellar extracts. MCAs 20H6, 2F4, 18G12 and 10C5 reacted with the lipopolysaccharide (LPS) of Xcc. MCAs 16B5 and 17C12 reacted with a 39 kilodalton and a 29 kilodalton protein, respectively.It is concluded that the PCAs and MCAs discussed in this study may be used for routine identification and differentiation of (a group of) Xcc strains. The significance of the cross-reactions with other pathovars ofX. campestris needs to be determined by testing seed lots.     

Signal interactions in pathogen and insect attack: systemic plant-mediated interactions between pathogens and herbivores of the tomato,Lycopersicon esculentum

Plant-mediated interactions (i.e., induced resistance) between plant pathogens and insect herbivores were investigated using several pests of the cultivated tomato,Lycopersicon esculentum. Single leaflets of tomato leaves were injured by allowing a third-instarHelicoverpa zealarva to feed on the leaflets or by inoculating the leaflets withPseudomonas syringaepv.tomato(the causal agent of bacterial speck in tomato;Pst) or withPhytophthora infestans(the causal agent of late blight). Leaflets on separate plants were sprayed with benzothiadiazole, a chemical inducer of resistance toPst. The effects of these treatments on the resistance of uninoculated or undamaged leaflets to bothPstandH. zeawere then assessed after appropriate periods of time. The levels or activities of several defense-related proteins were determined in parallel. Infection of leaflets byPstdecreased the suitability of uninoculated leaflets of the same leaf for bothH. zeaand forPst. Similarly, feeding byH. zeacaused leaf-systemic increases in resistance to bothH. zeaandPst. Infection of leaflets byP. infestans, in contrast, had no effect on resistance of leaflets toH. zea. Treatment of leaves with benzothiadiazole induced resistance toPstbut improved suitability of leaflets forH. zea. Feeding byH. zeacaused the systemic accumulation of proteinase inhibitor mRNA and the systemic induction of polyphenol oxidase activity; in contrast, treatment with benzothiadiazole and inoculation withP. infestanscaused the systemic accumulation of pathogenesis-related protein mRNA and the systemic induction of peroxidase activity. Inoculation of leaflets withPstcaused the leaf-systemic accumulation of both pathogenesis-related protein and proteinase inhibitor mRNA and the systemic induction of both peroxidase and polyphenol oxidase activity. These results provide clear evidence for reciprocal induced resistance involving certain pathogens and arthropod herbivores of tomato. In addition, these results provide several insights into the integration and coordination of the induced defenses of tomato against multiple pests and suggest that the expression of resistance against some pests may compromise resistance to others.     

Local and systemic resistance to fungal pathogens triggered by an AVR9-mediated hypersensitive response in tomato and oilseed rape carrying the Cf-9 resistance gene

Tomato and transgenic oilseed rape plants expressing the Cf-9 resistance gene develop a hypersensitive response (HR) after injection of the corresponding Avr9 gene product. It was investigated whether induction of a HR conferred resistance to different fungal pathogens in tomato and oilseed rape. Induction of an AVR9 mediated HR at the pathogen infection site delayed the development of the biotrophs Oidium lycopersicum in tomato and Erysiphe polygoni in oilseed rape, but enhanced the development of the necrotrophs Botrytis cinerea and Alternaria solani in tomato and Sclerotinia sclerotiorum in oilseed rape. Interestingly, delayed fungal disease development was observed in plant tissues surrounding the HR lesion regardless of whether a necrotrophic or biotrophic pathogen was used. In tomato, AVR9 injection induced systemic expression of PR1, PR2 and PR3 defence genes but did not induce systemic resistance to O. lycopersicum, B. cinerea or A. solani. In oilseed rape, AVR9 injection temporarily induced systemic resistance to Leptosphaeria maculans and E. polygoni, but did not induce detectable systemic expression of PR1, PR2 or Cxc750. These results give new insights into the potential uses of an induced HR to engineer disease resistance.