Induced resistance

Plants can respond to pathogen infection by rapidly activating pre-existing defense mechanisms (induced resistance). An initial stimulus is required to bring about persistent systemic biochemical changes in the plant. Upon subsequent fungal, bacterial, or viral infection, these changes will result in the rapid and intense expression of disease resistance. Various agents capable of inducing plant defense mechanisms have already been described, e.g., chemicals, metabolic products of infection agents, non-pathogens of a host, and isolates of a pathogen that are not pathogenic to a certain host cultivar/clone. Inducible defense mechanisms are advantageous to the plant, since accumulation of phytotoxic compounds only occurs when necessary. No energy or metabolites are used in the absence of infection {[832]}.

Induction of resistance responses may be used as a tool to control plant disease. As presented in Table 20, suppression of wilt symptoms and induction of DED resistance can be achieved by pre-inoculation of the elm tree with non-pathogens as well as with non-aggressive isolates of O. ulmi s.l. An important prerequisite for this approach is that trees are treated annually before infection takes place. Curative treatment generally fails {[621],[627],[825]}. The effectiveness of the induced resistance approach varies considerably among the various elm clones/species. In addition, the method used for preventative inoculation affects the level of protection against DED {[625],[825]}. The induction of host defense mechanisms by pre-inoculation does not offer protection against root-graft-mediated O. ulmi s.l. infection {[625]}.

Non-aggressive isolates of O. ulmi s.l. suppress DED symptom development in elms susceptible to O. novo-ulmi {[621]}. However, pre-inoculations of the elm clone ‘Lobel' with the non-aggressive DED fungusonly slightly enhances the tree's moderate resistance to O. novo-ulmi {[89]}. Possibly, specific DED defense mechanisms, which are more or less specifically triggered by a certain pathogen, can act simultaneously in elm.

Table 20:        Reduction of O. novo-ulmi-induced DED symptom expression in various elms after preventative inoculation with several bacteria and fungi {[89],[107],[ 621],[622],[625],[627],[628],[638],[639],[ 825],[829],[830],[836],[846]}

¹              not pathogenicfor elm

²              race EAN with an unusual low virulence on elm

³              different isolates that belong to the OPH (O. piceae hardwood) population that attacks only hardwood hosts{[89],[107]}

⁴           different isolates tested

⁵              isolate virulent on tomato and potato

⁶           treatment with this fungus alone results in severe wilting of the elm after 10 days

⁷              also no disease suppression in the hybrid Huntingdon elm {[829]}

⁸              U. x hollandica Vegeta x U. minor

⁹              (U. glabra Exoniensis x wallichiana) x (U. hollandica Bea Schwarz selfed)

n.d.      not determined

+         pre-inoculation with this microorganism results in reduction of elm wilt symptoms

+/-       variable results

-           no reduction of wilt symptoms

*          Ricard {[830]} reports the biocontrol of DED in non-specified elm species/clones by preventative inoculation with the Trichoderma viride isolates IMI206039 and IMI 206040

Apparently, the induction of the less pathogen-specific host response in such O. novo-ulmi-susceptible elms as ‘Commelin'is enough to obtain a sufficient level of DED resistance. Hubbes and Jeng {[827]} suggest that U. americana can be protected against DED by preventative inoculation with the non-aggressive O. ulmi isolate 311 applied at a concentration of 2.5 10⁵ spores/ml. However, the scale of their experiments was too small to be conclusive.  

A number of the bacteria and fungi used for pre-inoculation of elm—such as Pseudomonas sp. and Trichoderma viride, and Phaeotheca dimorphospora—exhibit antagonistic activity against O. ulmi s.l. (Photo 60, see Enemies of O. ulmi s.l.). The Deuteromycete P. dimorphospora shows a strong antibiotic activity against O. ulmi and O. novo-ulmi in vitro {[213]}. However, althoughpre-inoculation of U. americana seedlings with this fungus results in protection against O. ulmi infection, no protection is provided against O. novo-ulmi infection {[119]}. Lanier {[846]} reported that injection of 15- to 30-year-old American elms with T. viride induces resistance against O. ulmi. The very limited zone of discoloration around the injection zones suggests that T. viride does not colonize the tree. Since the fungus T. viride does not synthesize antimycotics, its mycoparasitic activity may be based on the production of cellulases, chitinases, and glucanases. The cell wall of O. ulmi contains large amounts of cellulosic b-(1.4) glucans {[834]}.

Pseudomonas fluorescens and P. syringae are known to produce antifungal metabolites {[153],[830]}. Strobel and Myers {[622]} reported that, in contrast to preventative treatment of elms with a wild type strain of P. syringae, non-antimycotic producing isolates of this fungus have no significant effect on DED development. However, experiments by Scheffer {[638]} suggest that DED resistance obtained after pre-inoculation with Pseudomonas sp. does not result from direct antibiosis between bacteria and fungus. In his study, four Pseudomonas isolates, varying from a low to a potent producer of antifungal compounds, were able to strongly suppress DED symptom development. Although the observations of Strobel and Myers {[622]} and Scheffer {[638]} appear to be contradictory, it should be noted that the production of antimycotics was analyzed using in vitro assays. Since data obtained in these assays do not necessarily reflect the natural situation, synthesis of antifungal metabolites by the various isolates in vivo has to be determined.

Photo 60:        Antagonism of O. ulmi with P.syringae (top left) and other Pseudomonas strains (lower panels). Control top right.       

Probably, induction of defense mechanisms is based on the recognition of specific signals, and will therefore not occur after inoculation of elms with every arbitrary microorganism. Apparently, the host plant recognizes and responds to specific signals. Non-pathogens like Ceratocystis narcissi and Verticillium albo-atrum V22W do not affect the progress of DED {[621]}. Experiments by Elgersma and Overeem {[715]} showed that a V. albo-atrum isolate from tomato does induce mansonone synthesis in elm. It remains to be determined whether or not the inability of V. albo-atrum V22W to induce DED resistance is based on its possible inability to induce mansonone synthesis in elm. Furthermore, the relation between high concentrations of phytoalexins and DED resistance is still unclear {[149],[308],[641],[715]}.

While such microorganisms as Ophiostoma piceae and Pseudomonas sp. are able to induce a sufficient level of host defense response in only a limited number of elm clones/species, preventative treatment of elm trees with the V. dahliae isolates WCS850 or WCS424 appears to be effective in protecting against DED all elms tested (Table 20, Photo 61, {[89],[625]}). V. dahliae is a vascular wilt pathogen with a worldwide host range of at least 136 plant species, including vegetables, legumes, fruit, nut trees, forest trees, and woody and herbaceous ornamentals {[837],[840],[841]}. Verticillium isolates from a given host cause a range of symptoms on other hosts. However, the fungus shows the highest pathogenicity towards the species from which it was obtained. Generally, the disease symptoms will be the most severe on this host {[837]}. Recently, Bianchini et al. {[838]} reported a rapid induction of sesquiterpenoid phytoalexin synthesis in resistant cultivars of cotton after infection with V. dahliae.

In contrast to the V. dahliae isolates WCS850 and WCS424, the isolates 324 and Vd-280 are not able to induce a sufficient level of resistance in elm. The reason for this discrepancy still has to be elucidated. Verticillium sp. are imperfect fungi that use the vegetative compatibility system for gene flow and recombination {[515]}. Vegetative incompatibility results in distinct gene pools (termed vegetative compatibility groups or VCGs) in a fungal species that may differ in many characteristics {[837]}. V. dahliae strains isolated from potato are often incorporated into VCG 4, and isolates derived from ornamental woody plants are primarily assigned to VCG 1 and VCG2 {[840],[841]}. While V. dahliae WCS 850 and WCS424 have been obtained from potatoand eggplant (S. melongena), the isolates 324 and Vd-280 were isolated from the trees Acer campestre and Olea europaea (D. Elgersma, personal communication, {[89]}). Certain V. dahliae strains have been isolated from elm {[841]}. Perhaps in contrast to the genetically more distinct strains obtained from Solanum sp., the tree-derivedisolates 324 and Vd-280 show a close relation to the V. dahliae fungi that are found in elm. As a result, the elm may fail to recognize them as microorganismsto which it needs to respond by induction of its defense mechanisms. In addition, the elm tree may even become diseased after infection with these fungi. The latter situation may explain the observed wilting of English elm after infection with V. dahliae 324 {[89]}.

Experiments by Sutherland et al. {[89]} indicate that suppression of DED symptom expression can only be obtained when pre-inoculations are performed with viable fungi, including their metabolites. Pretreatments with chitin or gamma-irradiated conidia of O. ulmi R21, O. novo-ulmi H106, V. dahliae 324, or O. piceae H1042 did not result in reduction of DED symptom expression. Recently, M. Hubbes patented an elicitor glycoprotein derived from the culture filtrate of the non-aggressive O. ulmi isolate Q412 (world patent WO98/43483). The isolated protein appears to have a molecular weight of 21 KD {[184]}. Beginning at the N-terminus, the elicitor starts with the amino acid sequences 1 and 2 given in 21. These fragments do not show homology to any published protein sequence. The amino acid sequence of two internal elicitor fragments (termed 3 and 4) are also presented in Table 21. Hubbes determined a part of the genomic DNA sequence of the gene encoding the elicitor. This 687 bp sequence encompasses the sequences encoding the amino acids of fragments 3 and 4 separated by 554 bp (Fig. 9). The gap between the 687 bp gDNA sequence and the portion of the gene encoding the N-terminal fragments 1 and 2 is thought to be approximately 200 bp. Figure 10 presents an overview of the current knowledge of the O. ulmi Q412 elicitor. The 687 bp gDNA sequence shows the greatest homology to aspartic

Table 21:        N-terminal and internal amino acid sequences from the glycoprotein elicitor isolated from O. ulmi Q412 culture filtrate (WO98/43483)

¹           Possible amino acids at certain positions are given in small letters

proteinases of the fungi Glomerella cingulata (65.6%), Cryphonectria parasitica (62.6%), Podospora anserian (62.5%), and Rhizopizes chinensis (51.8%) {[843],[844],[845],[853]}. Aspartic proteinases belong to an important family of hydrolytic enzymes found in animals and plants. These proteins are associated with a variety of biological processes, such as hypertension, muscular dystrophy, and programmed cell death. Therefore, they may have a more general function in stress signaling rather than being specific to one particular process. Endothiapepsin—an aspartic proteinase secreted by C. parasitica—is used on a commercial scale as a source of milk-clotting enzyme for cheese-making {[845]}. The active site of aspartic proteinase consists of two carboxyl groups and a tightly bound water molecule. Possibly, the reaction of this protein with certain elm components results in the release of more specific inducers of host defense responses.

Fig. 9:              687 bp genomic DNA sequence of the glycoprotein  elicitor of O. ulmi isolate Q412 (WO98/43483)

^III             gDNA fragment encoding the internal amino acid fragment 3

^IV             gDNA fragment encoding the internal amino acid fragment 4

Fig.10:            Overview of the current knowledge concerning the elicitor of O. ulmi Q412 Q412 (WO98/43483)

The amino acid sequences that have been analyzed are presented as gray boxes. Fragments I and II belong to the N-terminus of the protein; fragments II and IV are internal fragments. The number of amino acids is given for each fragment separately. Fragment V comprises 687 bp of the gDNA sequence encoding the elicitor. The gap between fragment V and the N-terminal sequence is thought to be approximately 200 bp. The gDNA and amino acid sequences are shown in Figure 9 and Table 21.