Effect of Harpin from Four Pathovars of Pseudomonas syringae on Pea Defense Responses

To investigate the role of the proteinaceous elicitor, harpin, on host and nonhost plants, we isolated the harpin-coding gene, hrpZ, from Pseudomonas syringae pvs. pisi, glycinea, tabaci and tomato. Effects of the recombinant harpin proteins on pea plants were analyzed and compared with the effects of the corresponding bacterial treatment. After inoculation of pea with pea pathogen P. syringae pv. pisi, the bacterial population increased and the accumulation of PAL-mRNA and pisatin was inhibited. The nonpathogenic pathovars, glycinea, tabaci and tomato induced both defense responses in pea. However, none of the harpins induced the hypersensitive reaction or accumulation of PAL-mRNA and pisatin in pea. Harpins from P. syringae pvs. glycinea, tomato and pisi did induce these defense responses in tobacco, however, suggesting that externally applied harpins either are not recognized or are nonfunctional in pea plants. Received 27 June 2000/ Accepted in revised form 21 February 2001     

Pisatin involvement in the variation of inhibition of Fusarium oxysporum f. sp. pisi spore germination by root exudates of Pisum spp. germplasm

Fusarium wilt caused by Fusarium oxysporum f. sp. pisi (Fop) is one of the major constraints of pea worldwide. Its control is difficult and is mainly based on the use of resistant cultivars. This study aimed to identify and characterize resistance mechanisms interfering with Fop spore germination, as an additional pre‐penetration resistance mechanism little explored so far. For this, root exudates were collected from 12 pea accessions with differential responses to the disease, from resistant to susceptible, and their effects on Fop germination and growth were determined. While root exudates from most accessions stimulated Fop germination, the root exudates of three accessions, JI 1412, JI 2480 and P42, did not stimulate, or even inhibited, Fop germination. Although some additional compounds might be involved, the analysis showed that the most active metabolite was the pea phytoalexin pisatin. Pisatin was identified in the active fraction of pea root exudate extracts and its amount in the root exudates was negatively correlated with the extent of Fop germination. This suggests an important role of pisatin in the constitutive defence of pea against F. oxysporum.     

Pisatin demethylation by fungal pathogens and nonpathogens of pea: association with pisatin tolerance and virulence

Previous studies have indicated that detoxification of their hosts' phytoalexins is a tolerance mechanism for some true fungi, but not the fungus-like Oomycota, and may be involved in determining the virulence of a pathogen. In the present study, the associations between demethylation of the pea phytoalexin pisatin, tolerance to pisatin, and virulence on pea were examined for 50 fungal isolates which represent 17 species of pathogens and nonpathogens of pea. All isolates ofPythium coloratum and P. irregulare failed to metabolize and were sensitive to pisatin, consistent with previous observations that members of the Oomycota generally lack the ability to metabolize and are sensitive to their hosts' phytoalexins. Among true fungi tested, the ability to demethylate pisatin was common, regardless of whether the particular isolate was pathogenic on pea or not. However, when the rate of pisatin demethylation was compared to virulence, all but one of the moderate to highly virulent isolates rapidly demethylated pisatin. In addition, the more rapidly demethylating isolates were generally more tolerant of pisatin. These results suggest that a specialized enzyme system for quickly detoxifying pisatin might be present in most pea pathogens. In previous studies a specific cytochrome P450 enzyme for demethylating pisatin was identified in the pea pathogen Nectria haematococca mating population VI, and genes (PDA genes) encoding that enzyme have been cloned from this fungus. When DNA specific for these genes was used to probe genomic DNA from other fungi that demethylate pisatin, significant hybridization was detected with only one fungus, the pea pathogen Fusarium oxysporum f. sp. pisi. If the other pea pathogens possess a specific cytochrome P450 system for detoxification of pisatin, the genes encoding these enzymes apparently share limited nucleotide similarity with N. haematococca PDA genes.     

Wheat leaf resistance to Pyrenophora tritici‐repentis induced by silicon activation of phenylpropanoid metabolism

Tan spot caused by Pyrenophora tritici‐repentis is a disease present in all wheat‐producing countries and silicon (Si) treatment of wheat plants has been shown to increase plant resistance to tan spot. In this study, the effect of phenylpropanoid metabolism on resistance to tan spot was evaluated and some phenolic compounds that accumulated in response to P. tritici‐repentis attack were identified. Furthermore, the effect of Si on phenylalanine ammonia‐lyase (PAL) activity and phenolic compound accumulation were determined in situ. Antifungal activity of differentially accumulated phenolic compounds was also evaluated in in vitro tests. Results showed that the increase in concentration of phenolic compounds was greatest at the onset of infection, and that some compounds showed fungitoxic effects including fungal tip swelling, granulation of germ tube and hyphae, and hyphal hyperbranching. Silicon‐induced reduction in both lesion size and tan spot disease progression were associated with activation of phenylpropanoid metabolism. PAL activity and accumulation of antifungal phenolic compounds were greater in pathogen‐inoculated plants supplied with Si. In these plants, fluorescence indicative of accumulation of phenolic compounds occurred early in epidermal cells and its intensity increased during the evaluation period, showing higher numbers of fluorescent cells around infected cells. Thus, the combined responses of cell fluorescence at sites of infection, increased PAL activity and accumulation of phenols indicate that Si strengthened wheat defence responses to infection by P. tritici‐repentis, reducing the severity of tan spot.     

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.     

Granular formulation of Fusarium oxysporum for biological control of faba bean and tomato Orobanche

BACKGROUND: Orobanche spp. represent a serious threat to a wide range of crops. They are difficult targets for herbicides, and biological control could provide a possible solution. This work therefore aimed to formulate mycoherbicides of Fusarium with adequate shelf life and virulence against Orobanche but safe to faba bean and tomato. RESULTS: Only two isolates of Fusarium oxysporum Schlecht. (Foxy I and Foxy II) obtained from diseased Orobanche shoots were found to be pathogenic to Orobanche crenata Forsk. and Orobanche ramosa L. Conidial suspension of both isolates significantly decreased germination, attachments and tubercles of Orobanche. Microconidia and chlamydospores of both isolates were formulated as mycoherbicides encapsulated in a wheat flour–kaolin matrix (four different formulations). All formulations greatly diminished Orobanche emerged shoots, total shoot number, shoot height, attachment of emerged shoots, the germinated seeds that succeeded in emerging above the soil surface and dry weight. Meanwhile, disease incidence and disease severity of emerged shoots were enhanced. The shelf life was adequate, particularly for coarse, freshly prepared, low‐temperature‐stored, microconidia‐rich formulations. The induced growth reduction of Orobanche‐infected host plants seemed to be nullified by formulations, particularly at the highest dose. CONCLUSION: These formulations seemed to destroy Orobanche but appeared harmless to host plants. Hence, they could be efficiently used as mycoherbicides for biological control of Orobanche in faba bean and tomato. Copyright © 2008 Society of Chemical Industry     

Bacterial induction of the accumulation of phaseollin,pisatin and rishitin and their antibacterial activity

Bean hypocotyls, pea pods and tomato fruits were tested for phaseollin, pisatin and rishitin production when challenged with the phytopathogenic bacteriaErwinia carotovora, Pseudomonas phaseolicola, P. pisi andP. solanacearum, and their isolated extracellular polysaccharides. All bacteria induced phytoalexin accumulation, whereas only phaseollin and pisatin, but not rishitin, were elicited by EPS. The inhibitory effect of these three phytoalexins on bacterial growth was studied in liquid medium; whereas phaseollin and pisatin strongly inhibited growth, only a slight inhibitory effect resulted from the presence of rishitin in the medium.Samenvatting Bonehypocotylen, erwtepeulen en tomatevruchten werden onderzocht op hun vermogen tot vorming van respectievelijk faseolline, pisatine en rishitine, na inoculatie met de fytopathogene bacteriënErwinia carotovora, Pseudomonas phaseolicola, P. pisi enP. solanacearum en na behandeling met oplossingen van hun extracellulaire polysacchariden (EPS). Alle bacteriesoorten induceerden fytoalexinevorming, terwijl hun EPS wel faseolline- en pisatine-, maar geen rishitinevorming induceerden. Faseolline en pisatine remden de groei van de bacteriën in vloeibaar medium sterk; rishitine daarentegen had slechts een geringe groeiremming tengevolge.     

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.     

Suppression of clubroot by Clonostachys rosea via antibiosis and induced host resistance

The mechanism of the biofungicide Prestop^® (Clonostachys rosea) was investigated for control of clubroot (Plasmodiophora brassicae) on canola. The key product components were partitioned and assessed for their effect on pathogen resting spores, root hair infection (RHI) and disease development using light microscopy, quantitative PCR and different application treatments during infection. The whole product of Prestop was consistently more effective than the C. rosea conidial suspension or product filtrate alone in reducing RHI and clubroot development. This biofungicide showed little effect on germination or viability of resting spores. Two‐application treatments at seeding and 7–14 days after seeding achieved greater clubroot control than a single application of the biofungicide at either seeding or post‐seeding stage. This may indicate the need to maintain a high biofungicide dose in the soil during primary and secondary infection. This biocontrol fungus colonized the rhizosphere and interior of canola roots extensively, and possibly induced plant resistance based on up‐regulation of the genes that are involved in jasmonic acid (BnOPR2), ethylene (BnACO) and phenylpropanoid (BnOPCL, BnCCR) biosynthetic pathways. It is concluded that the biofungicide Prestop suppressed clubroot on canola at least via root colonization and induced systemic resistance (ISR), and the latter may be through the modulation of phenylpropanoid and jasmonic acid/ethylene metabolic pathways elicited by the fungus.     

Implication of peroxynitrite in defence responses of potato to Phytophthora infestans

In this study peroxynitrite (ONOO^?) is proposed as an important player in defence responses during the interaction of potato (Solanum tuberosum) and the oomycete pathogen Phytophthora infestans. The potato–avr P. infestans model system exhibited a transient programme of boosted ONOO^? formation correlated in time with the burst of nitric oxide (NO) and superoxide during the first 6 h post‐inoculation (hpi). The early ONOO^? over‐accumulation was not accompanied by TPx gene expression. In contrast, the compatible interaction revealed a 24 h delay of ONOO^? formation; however, an enhanced level of NO and superoxide correlated with TPx up‐regulation was recorded within the earlier stages of pathogen infection. Peroxynitrite over‐accumulation in the susceptible potato coincided with an enhanced level of protein tyrosine nitration starting from 24 hpi. Surprisingly, the nitroproteome profile of the resistant potato did not show any visible difference after inoculation, apart from one band containing subtilisin‐like protease‐like proteins, which appeared 48 h after pathogen attack. An additional pharmacological approach showed that treatment of the susceptible genotype with ONOO^? followed by inoculation with P. infestans contributed to slowing down of the colonization of host tissues by the pathogen via a faster and stronger up‐regulation of the key defence markers, including the PR‐1 gene. Taken together, the results obtained indicate that a precise control of emitted NO and superoxide in cooperation with thioredoxin‐dependent redox sensors in sites of pathogen ingress could generate a sufficient threshold of ONOO^?, triggering defence responses.     

Genetic diversity of Moroccan populations of Orobanche foetida: evolving from parasitising wild hosts to crop plants

Orobanche foetida is widely distributed in western Mediterranean countries infecting wild legumes. Recently it has been reported in Morocco infecting common vetch, presenting a further constraint for legume production in this area. Comparative studies between Orobanche populations attacking wild species and Orobanche populations growing on crops from the same region may help to clarify whether a host specialisation process occurred. In the present study, the genetic variability of five O. foetida populations, four infecting wild plants and one infecting cultivated vetch in Morocco was characterised using amplified fragment‐length polymorphism (AFLP) markers to elucidate the existing genetic relationship between populations and to suggest a potential origin for the recently detected vetch‐infecting population. Analysis of molecular variance suggested the existence of genotypic differentiation among populations. The most genetic divergent population by cluster analysis was the population collected on Ornithopus sativus. The vetch‐infecting O. foetida population was closer to the three populations infecting Scorpiurus muricatus. The possibility of the presence of a reservoir of diversity is balanced against direct genetic adaptation and completely new introduction. The potential for this Orobanche species to shift host and become a threat to agriculture exists and has to be taken into account for future legume breeding for these areas.     

Physcion,a natural anthraquinone derivative,enhances the gene expression of leaf‐specific thionin of barley against Blumeria graminis

BACKGROUND: Physcion is a key active ingredient of the ethanol extract from roots of Chinese rhubarb (Rheum officinale Baill.) that has been commercialised in China for controlling powdery mildews. The biological mechanism of action of physcion against the barley powdery mildew pathogen was studied using bioassay and microarray methods. RESULTS: Bioassay indicated that physcion did not directly affect conidial germination of Blumeria graminis Speer f. sp. hordei Marchal, but significantly inhibited conidial germination in vivo. Challenge inoculation indicated that physcion induced localised resistance rather than systemic resistance against powdery mildew. Gene expression profiling of physcion‐treated barley leaves detected four upregulated and five downregulated genes (ratio ≥ 2.0 and P‐value < 0.05) by using an Affymetrix Barley GeneChip. The five upregulated probe sequences blasted to the same barley leaf‐specific thionin gene, with significant changes varying from 4.26 to 19.91‐fold. All downregulated genes were defence‐related, linked to peroxidase, oxalate oxidase, bsi1 protein and a pathogenesis‐related protein. These changes varied from ? 2.34 to ? 2.96. Quantitative real‐time PCR data confirmed that physcion enhanced the gene expression of leaf‐specific thionin of barley. CONCLUSION: Results indicated that physcion controls powdery mildew mainly through changing the expression of defence‐related genes, and especially enhancing expression of leaf‐specific thionin in barley leaves. Copyright © 2010 Society of Chemical Industry     

Inverse effects of Arabidopsis NPR1 gene on fusarium seedling blight and fusarium head blight in transgenic wheat

In this study, the Arabidopsis thaliana NPR1 (non‐expressor of PR genes) gene was integrated into an elite wheat cultivar, and the response of the transgenic wheat expressing NPR1 to inoculation with Fusarium asiaticum was analysed. With seedling inoculation, the transgenic lines showed significantly increased fusarium seedling blight (FSB) susceptibility, whereas floret inoculation resulted in enhanced fusarium head blight (FHB) resistance. Quantitative real‐time PCR revealed that expression of two defence genes, PR3 and PR5, was associated with susceptible reactions to FSB and FHB, whereas the PR1 gene was activated in resistance responses. This inverse modulation by the constitutively expressed NPR1 gene suggests that NPR1 has a bifunctional role in regulating defence responses in plants. Therefore, it is unsuitable for improving overall resistance to FSB and FHB in wheat.     

Importance of AC-rich Element on Pea Phenylalanine Ammonia-Lyase Gene 1 Promoter for Expression Induced by Nonpathogenic Attack

Regulatory elements in the promoter of phenylalanine ammonia-lyase gene 1 of pea (PSPAL1) in response to nonpathogenic attack were identified by in vivo footprinting analysis. The footprints determined AC-rich sequences, Box-I and Box-II, that were conserved at similar positions in the phenylpropanoid gene promoters from several plants. To reveal the functions of the AC-rich sequence in nonpathogen-responsiveness, we constructed Box-I-deletion PSPAL1 promoter (dB-1) with GUS reporter gene and transformed it into tobacco plant. The dB-1 had reduced basal expression and a complete loss of nonpathogen-responsiveness. These results indicate the essentiality of Box-I for PSPAL1 activation induced by nonpathogenic attack. Received 27 October 1999/ Accepted in revised form 25 November 1999     

Programmed cell death pathways induced by early plant‐virus infection are determined by isolate virulence and stage of infection

Programmed cell death (PCD) pathways caused by Turnip mosaic virus (TuMV) infection before symptom appearance were studied by light microscopy and electrolyte leakage following sap inoculation of Brassica carinata (Ethiopian mustard) TZ‐SMN‐44‐6 plants. Leaf responses to inoculation with avirulent (TuMV‐avir) and virulent (TuMV‐vir) isolates, and mock‐inoculation, were compared at 2, 20 and 52 h after inoculation (hai). The phenotypes induced were localized resistance (TuMV‐avir) and systemic susceptibility (TuMV‐vir). No visible TuMV symptoms were recorded in any inoculated plants during the 2–52 hai sampling period, but appeared as chlorotic spots in inoculated leaves at 5 days after inoculation. With TuMV‐vir alone, they were followed by systemic infection (mosaic). Dead cell number, deformation, percentage area and percentage integrated intensity, and conductivity of electrolyte leakage data, were analysed to examine their possible roles in stimulating cell death pathways. At 2 hai, dead cell number and percentage area were significantly greater for TuMV‐avir than TuMV‐vir infection or mock‐inoculation. Overall, isolate TuMV‐vir caused significantly greater cell deformation than TuMV‐avir, whereas wounding by mock‐inoculation had negligible effects. By 52 hai, isolate TuMV‐avir caused significantly greater electrolyte leakage than isolate TuMV‐vir or mock‐inoculation. This suggests both isolates triggered morphological changes consistent with apoptotic‐like PCD and necrosis‐like PCD that depended upon isolate virulence and stage of infection, respectively. These findings highlight how quantification of dead cell deformation and electrolyte leakage offer a new understanding of compatible and incompatible plant responses to early virus infection in plants.     

Pathways of bacterial invasion and watermelon seed infection by Acidovorax citrulli

This study explored the pathways of ingress of Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbits, into watermelon seeds. Up until 7 days post‐inoculation (DPI), a significantly higher percentage of watermelon seeds was infected with A. citrulli when the bacteria were applied (c. 1 × 10⁶ colony‐forming units) to stigmas versus ovary pericarps of female flowers. Immunofluorescence microscopy showed that, with stigma inoculation, A. citrulli colonized style and ovary tissues by 1 DPI, and the bacteria co‐localized with pollen germ tubes in these tissues. With ovary pericarp inoculation, A. citrulli cells penetrated the epicarp and mesocarp tissues by 1 DPI but did not reach endocarp until 4 DPI. Finally, manual pollination followed by stigma inoculation led to >53% A. citrulli‐infected seed lots, while A. citrulli was not detected in seeds/ovules generated by stigma inoculation without pollination (chemically induced parthenocarpy). These results show that stigma inoculation results in faster colonization of watermelon ovules by A. citrulli than pericarp inoculation, even though there is no difference in the levels of infection in mature seeds. The data also indicate that pollen germ tubes play an important role in A. citrulli ingress into watermelon seeds via stigmas.     

Soyasapogenol B and trans-22-dehydrocam- pesterol from common vetch (Vicia sativa L.) root exudates stimulate broomrape seed germination

BACKGROUND: Orobanche and Phelipanche species (the broomrapes) are root parasitic plants, some of which represent serious weed problems causing severe yield losses on important crops. Control strategies have largely focused on agronomic practices, resistant crop varieties and herbicides, albeit with marginal success. An alternative control method is the induction of suicidal seed germination with natural substances isolated from root exudates of host and non‐host plants. RESULTS: Soyasapogenol B [olean‐12‐ene‐3,22,24‐triol(3β,4β,22β)] and trans‐22‐dehydrocampesterol [(ergosta‐5,22‐dien‐3‐ol, (3β,22E,24S)] were isolated from Vicia sativa root exudates. They were identified by comparing their spectroscopic and optical properties with those reported in the literature. Soyasapogenol B was very specific, stimulating the germination of O. minor seeds only, whereas trans‐22‐dehydrocampesterol stimulated P. aegyptiaca, O. crenata, O. foetida and O. minor. CONCLUSION: Soyasapogenol B and trans‐22‐deydrocampesterol were isolated for the first time from Vicia sativa root exudates, and their biological activity as stimulants of Orobanche or Phelipanche sp. seed germination was reported. Copyright © 2011 Society of Chemical Industry