Possible peroxidase functions in the interaction between the parasitic plant, Orobanche aegyptiaca, and its host, Arabidopsis thaliana

The mechanism by which the flowering holoparasitic plant, Orobanche aegyptiaca , infects its host without evoking a defence mechanism is still poorly understood. In this work, we studied several mechanisms used by phytopathogenic fungi. We focussed on the possible role of peroxidases during O. aegyptiaca penetration into the roots of Arabidopsis thaliana . A convenient experimental system for studying the interaction under sterile conditions was developed. The formation of extracellular reactive oxygen species (ROS) was detected at the interaction site before, during, and after the parasite penetrated into the host. These extracellular ROS probably originated from the parasite. However, no intracellular ROS could be detected at the site of the interaction. Peroxidase activity was observed mainly at the apex of the root of the parasite and in the adventitious roots of the tubercle. Benzhydroxamic acid, a peroxidase inhibitor, was used to probe the possible role of peroxidase in the infection process. Peroxidase activity was observed in the root apex and adventitious roots of O. aegyptiaca, but no evidence was found for its participation in the actual infection process. Peroxidase activity was also found in the later stages of the interaction between the host and the parasite. We propose that peroxidases could have a role in generating extracellular ROS for loosening the cell wall of the host in order to facilitate penetration. Alternatively, the ROS could act in facilitating the root elongation of the parasite.     

Some compatible Rhizobium leguminosarum strains in peas decrease infections when parasitised by Orobanche crenata

Orobanche crenata (crenate broomrape) produces serious damage to many legume crops and particularly becomes a limiting factor for pea production in the Mediterranean basin. Nodulation effects on pea–broomrape relationships were studied using the commercial pea cultivar Douce de Provence and different Rhizobium strains using pot and Petri dish experiments. First, the benefit of bacterial inoculation on plant growth and efficiency of N incorporation were demonstrated for two isolates, P.SOM and P.1236. These isolates did not influence parasite germination induced by the artificial stimulant, GR24. In contrast, pea root inoculation with P.SOM and P.1236 isolates led to a reduced root infection by O. crenata , resulting from a lower Orobanche germination rate close to pea roots and a limited capacity of the parasitic seedlings to develop tubercles. Broomrape necrosis was observed both before and after parasite attachment to inoculated pea roots. Concomitantly, reduction in infection was accompanied by enhanced peroxidase activity and constantly high phenylalanine ammonia lyase activity in pea roots. These data suggest the involvement of these enzymes in pea resistance to crenate broomrape induced by the compatible rhizobia. Management of Orobanche via crop selection based on these enzyme systems is a viable option.     

Possible function of isoleucine in the methyl jasmonate response of <Emphasis Type="Italic">Arabidopsis</Emphasis> to <Emphasis Type="Italic">Phelipanche aegyptiaca</Emphasis>

Resistance of Arabidopsis thaliana to Phelipanche aegyptiaca (syn. Orobanche aegyptiaca) can be induced by exposure of the host to low concentrations of gaseous methyl jasmonate, in a concentration-dependent fashion. Application of methyl jasmonate at marginal concentrations, 10⁻⁸ M, for 12 h reduced infection by 50%. However, if the host plants were supplied with isoleucine at 10⁻³ M, and the isoleucine removed prior to exposure to methyl jasmonate, infection was reduced to less than 20%. The presence of isoleucine throughout the experiment—before, during and after exposure to methyl jasmonate—reduced infection of the host to almost zero. These results suggest that when Arabidopsis is exposed to methyl jasmonate, full activation of the defense mechanisms, including formation of the isoleucine jasmonic acid conjugate, might be limited by the availability of isoleucine in the host. These findings add another factor to the complex interaction between infecting parasite, and evocation of host defense mechanisms.     

Identification of sources of resistance to crenate broomrape (Orobanche crenata) in Spanish lentil (Lens culinaris) germplasm

The weedy root parasite Orobanche crenata constitutes a serious threat to lentils and other legumes grown in the Mediterranean and western Asia. Control strategies have centred on agronomic practices and the use of herbicides. Resistance breeding is hampered by scarcity of proper sources of resistance and of a reliable and practical screening procedure. A stepwise procedure was used here. A Spanish germplasm collection of 234 accessions of lentils (Lens culinaris) was screened for resistance to crenate broomrape under field conditions. A wide range of responses was observed, however, complete resistance was not detected. Some accessions exhibited a substantial reduction in broomrape infection. Thirty‐five lentil accessions with reduced broomrape infection were selected for further screening in the field and to determine the components of resistance in pot and in mini‐rhizotron experiments. Low infection seemed to be based on a combination of various escape and resistance mechanisms: (i) a lower root density, (ii) a lower induction of Orobanche seed germination, (iii) the reduced establishment of broomrape radicles and (iv) a limited development of established tubercles. In addition, necrosis of tubercles was observed in some accessions. This study shows how complementary in vitro screening methods may be used to rank and identify lentil accessions with potential broomrape resistance, based on different resistance mechanisms which may in the future be combined into a single cultivar by breeding.     

Strigolactone deficiency confers resistance in tomato line SL-ORT1 to the parasitic weeds Phelipanche and Orobanche spp

The parasitic flowering plants of the genera Orobanche and Phelipanche (broomrape species) are obligatory chlorophyll-lacking root-parasitic weeds that infect dicotyledonous plants and cause heavy economic losses in a wide variety of plant species in warm-temperate and subtropical regions. One of the most effective strategies for broomrape control is crop breeding for broomrape resistance. Previous efforts to find natural broomrape-resistant tomato (Solanum lycopersicon) genotypes were unsuccessful, and no broomrape resistance was found in any wild tomato species. Recently, however, the fast-neutron-mutagenized tomato mutant SL-ORT1 was found to be highly resistant to various Phelipanche and Orobanche spp. Nevertheless, SL-ORT1 plants were parasitized by Phelipanche aegyptiaca if grown in pots together with the susceptible tomato cv. M-82. In the present study, no toxic activity or inhibition of Phelipanche seed germination could be detected in the SL-ORT1 root extracts. SL-ORT1 roots did not induce Phelipanche seed germination in pots but they were parasitized, at the same level as M-82, after application of the synthetic germination stimulant GR24 to the rhizosphere. Whereas liquid chromatography coupled to tandem mass spectrometry analysis of root exudates of M-82 revealed the presence of the strigolactones orobanchol, solanacol, and didehydro-orobanchol isomer, these compounds were not found in the exudates of SL-ORT1. It can be concluded that SL-ORT1 resistance results from its inability to produce and secrete natural germination stimulants to the rhizosphere.     

Variation in resistance to Orobanche crenata in species of Cicer

Crenate broomrape (Orobanche crenata) is a major constraint for legume cultivation in Mediterranean agriculture. Field trials, pot and in vitro experiments demonstrated that resistance to O. crenata is present in chickpea and wild species of Cicer. The resistance is the result of the combination of several mechanisms, including low induction of parasite seed germination and in some accessions, either a darkening at the infection site on the host root that prevents establishment, or a reduced development of established parasite tubercles.     

Volatile 1-octen-3-ol induces a defensive response in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

1-Octen-3-ol is a major volatile metabolite produced by mold fungi. When Arabidopsis plants were treated with 1-octen-3-ol, some defense genes that are turned on by wounding or ethylene/jasmonic acid signaling were induced. The treatment also enhanced resistance of the plant against Botrytis cinerea. When the induction of defense genes with 1-octen-3-ol was compared with that by volatile methyl jasmonate (MeJA) and methyl salicylate treatments, the induction pattern was similar to that caused by MeJA. Thus, Arabidopsis seems to recognize 1-octen-3-ol and consequently activates its defense response.     

列当(Orobanche spp. and Phelipanche spp.)种子的采集与预处理方法

列当(Orobanche spp.and Phelipanche spp.)是一种在世界范围内危害严重的根寄生杂草,防除列当的研究也越来越多。正确地采集和预培养列当种子是获得正确防除列当研究结果的前提。列出了大田正确采集列当种子的方法,以及对采集到的种子进行过筛提纯、加洗洁精和吐温20清洗后,再表面消毒和预培养,并通过发芽试验检验此方法的可行性。结果表明,经过处理的向日葵列当、瓜列当种子的发芽率最高分别为54.4%、59.1%。此方法可以应用于列当的采集和预处理过程中,可为列当研究和防除提供参考。     

Growth environment and seedling age affect constitutive and inducible defence in radiata pine

There is increasing interest in induced resistance for crop protection. This study investigated effects of growth environment on inducible defences in developing radiata pine (Pinus radiata) seedlings. Seedlings were grown in the presence or absence of Trichoderma in either a temperature-controlled growth room or in a greenhouse. After 3, 5 or 7 months, seedlings were sprayed with 2.25 mm methyl jasmonate (MJ), 1 week before quantification of monoterpenes and total phenolics. The magnitude of induction of monoterpenes and total phenolics by MJ was inversely proportional to constitutive content, was greater in the greenhouse than the growth room and was greater at 3 months than at 7 months. This may indicate a cost-saving strategy to limit defence induction once constitutive defence reaches a certain threshold. Growth room seedlings expressed greater constitutive resistance to terminal crook (Colletotrichum acutatum) and to diplodia dieback (Diplodia sapinea) than greenhouse plants, consistent with the relative differences in constitutive defence chemistry. Indeed, while MJ induced resistance to diplodia dieback in both environments, there was no difference in terminal crook incidence (4.1%) between MJ-treated and untreated seedlings in the growth room. By the end of the study, growth room seedlings had c. 70% more β-pinene in stems and 140% more total phenolics in needles than greenhouse plants but were 40% smaller, indicating a defence–growth trade-off. Trichoderma did not have a significant effect on defence chemistry or on pathogen resistance. These results demonstrate the potential to manipulate defence in radiata pine by modification of the growth environment.     

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.     

Evaluation of the pathogenicity of microorganisms isolated from Egyptian broomrape (Orobanche aegyptiaca) in Israel

Diseased Egyptian broomrape ( Orobanche aegyptiaca ) inflorescences were collected from a heavily broomrape-infested tomato ( Lycopersicon esculentum ) field in Israel. The microorganisms that were isolated from the diseased inflorescences were passed through Koch's postulates on Egyptian broomrape-parasitizing tomato roots in a polyethylene bag system and pots under greenhouse conditions. The fungi, Alternaria alternata , Macrophomina phaseolina , Rhizoctonia solani , and Fusarium solani , and the bacterium, Bacillus sp., were newly isolated from the diseased inflorescences of Egyptian broomrape and were found to be pathogenic to the parasite. Fusarium solani damaged all of the developmental stages of broomrape and prevented the damage that Egyptian broomrape causes to tomato plants. The level of pathogenicity and the damage of M. phaseolina , A. alternata , and Bacillus sp. to Egyptian broomrape in greenhouse experiments were relatively low. All the tested microorganisms are known as pathogens of tomato, yet none caused disease symptoms on the tomato plants grown in the inoculated polyethylene bags or in the pots. Fusarium solani demonstrated the highest potential for further development as a mycoherbicide for Egyptian broomrape control in tomato.     

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.     

Biochemical responses to ultraviolet‐C radiation and methyl jasmonate in Pinus radiata seedlings that accompany induced resistance to Diplodia pinea

Irradiation of Pinus radiata seedlings with ultraviolet‐C (UV‐C) radiation or spray application of methyl jasmonate (MeJA) each resulted in induced resistance to subsequent wound inoculation with Diplodia pinea. Induced resistance was expressed by lower incidence of disease and by reduced size of stem lesions than in untreated seedlings. UV‐C was more effective than MeJA and the induced resistance was greatest in seedlings that were irradiated with UV‐C for 60 min, 1 week before pathogen inoculation. Induced resistance in the UV‐C treated stems was concomitant with increases in peroxidase (POX) and polyphenol oxidase (PPO) activity but showed no correlation with concentrations of α‐pinene and β‐pinene or total phenolics. Furthermore, POX induction in stem tissue was cumulative and was greatest after three repeat treatments with UV‐C at 6, 3 and 1 week before inoculation. In contrast to UV‐C, MeJA induced a significant increase in β‐pinene concentration in stem tissue but did not affect PPO activity. POX activity was induced by MeJA in stems, although to a lesser extent than by UV‐C, but was not affected in needles. This appears to be the first report demonstrating the use of UV‐C radiation to induce resistance to fungal infection in a coniferous species. The implications of the underlying differences between UV‐C‐ and MeJA‐mediated resistance to D. pinea are discussed.     

茉莉酸甲酯对水稻白叶枯病的诱导抗性及相关防御酶活性的影响

为探索茉莉酸甲酯(methyl jasmonate,MeJA)诱导水稻抗白叶枯病的效应,采用MeJA喷雾处理剪叶接种法,测定MeJA对水稻幼苗的白叶枯病病情指数、白叶枯病病菌Xanthomonas oryzae pv.oryzae的抑菌效果及对叶片过氧化物酶(peroridase,POD)、过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)、多酚氧化酶(polyphenol oxidase,PPO)和苯丙氨酸解氨酶(phenylalnaine ammonialyase,PAL)等相关防御酶活性的影响.0.05 ～ 2.0 mmol/L的MeJA能降低水稻幼苗白叶枯病的病情指数,但对水稻白叶枯病菌无直接抑菌活性;0.1 mmol/L MeJA的诱导效果最好,处理48h后,感病品种温229和抗病品种嘉早312的诱导效果分别为73.18％和70.43％;0.05 ～2.0mmol/L的MeJA处理水稻叶片中POD、CAT、SOD、PPO和PAL活性呈上升趋势.研究表明MeJA能诱导水稻幼苗对白叶枯病的抗性,且诱导抗性的产生与MeJA提高水稻相关防御酶的活性有关.     

Molecular analysis of resistance mechanisms to Orobanche cumana in sunflower

Resistance to the dicotyledenous parasite Orobanche cumana in sunflower is characterized by a low number of parasitic attachments and a confinement of the parasite in host tissues leading to its necrosis. To help understand what determines such resistance mechanisms, molecular, biochemical and histological approaches were employed before (early response) and after (late response) attachment of the broomrape parasite to susceptible (2603) and resistant (LR1) sunflower genotypes. The expression patterns of 11 defence-related genes known to be involved in different metabolic pathways (phenylpropanoids, jasmonate, ethylene) and/or in resistance mechanisms against microorganisms were investigated. RT-PCR and cDNA blot experiments revealed that the resistant genotype exhibited a stronger overall defence response against O. cumana than the susceptible one, involving marker genes of the jasmonate (JA) and salicylic acid (SA) pathways. Among them, the SA-responsive gene, def. (defensin), appeared to be characteristic of LR1 sunflower resistance. However, no JA accumulation and similar SA contents (250–300 ng g⁻¹ FW) were measured by GC/MS in both genotypes, parasitized or not. In addition, three cDNAs, isolated by a suppression-subtractive hybridization, were shown to be strongly induced only in the resistant genotype 8 days post-inoculation, when the first O. cumana attachments occurred. These genes, putatively encoding a methionine synthase, a glutathione S-transferase and a quinone oxidoreductase, might be involved in detoxification of reactive oxygen species, suggesting the occurrence of an oxidative burst during the incompatible interaction. Finally, host cell-wall modifications leading to parasite-confinement were correlated with more intense callose depositions in the resistant genotype, concomitant with over-expression of the callose synthase cDNA HaGSL1 .     

Inducible Flavone in Oats (Avena sativa) Is a Novel Defense Against Plant-Parasitic Nematodes

ABSTRACT The induction of defense compounds in oats (Avena sativa) in response to invasion by parasitic nematodes and to application of the wound hormone methyl jasmonate was examined. Oats cv. Quoll seedlings were challenged with Pratylenchus neglectus, Heterodera avenae, and Ditylenchus dipsaci and treated with 1 x 10(-4) M methyl jasmonate. Three compounds, isolated in methanolic root and shoot extracts of oats, exhibiting an absorbance spectrum typical of flavone glycosides, were induced by nematode invasion and methyl jasmonate. These were identified as flavone-C-glycosides by mass spectrometry. The effect of the flavone-C-glycosides on the invasion by and development of cereal cyst nematode H. avenae was assessed using methanolic extracts of shoots and roots from methyl jasmonate-treated plants. Both extracts impaired nematode invasion and development. When the extracts were fractionated by high voltage paper electrophoresis, only one flavone-C-glycoside, O-methyl-apigenin-C-deoxyhexoside-O-hexoside, inhibited nematode invasion. The protective effect of the induction of flavone-C-glycosides in oats by methyl jasmonate was evaluated against H. avenae and P. neglectus. Treatment with methyl jasmonate reduced invasion of both nematodes and increased plant mass, compensating for damage caused by the nematodes, and is attributed to the active flavone-C-glycoside. The active compound, O-methyl-apigenin-C-deoxyhexoside-O-hexoside, has not been implicated previously in plant defense against any pest or pathogen, and appears to provide protection against the major cereal nematodes Heterodera and Pratylenchus.     

THE EFFECT OF SUPERPHOSPHATE APPLICATION, 2,4-DB AND GRAZING ON BROOMRAPE (OROBANCHE MINOR SM.) IN A SUBTERRANEAN CLOVER PASTURE

Two 5-year trials were conducted in New South Wales on the control of broomrape (Orobanche minor Sm.), a plant parasite of subterranean clover (Trifolium subterraneum L.) and skeleton weed (Chondrilla juncea L.). Applications of superphosphate to two soils of low phosphorus status stimulated clover growth, which caused a reduction in the number of skeleton weed plants and a decrease in the broomrape population. Similar but less effective control was obtained when subterranean clover pasture was sprayed with 2,4-DB for skeleton weed control as soon as the clover seedlings had developed trifoliate leaves. In spring, sheep readily grazed the broomrape stems before flowering, causing death of the plants. Autumn application of super phosphate, coupled with grazing of the pasture by sheep, significantly reduced broomrape density.