Lorne R. Adam

Chitosan in plant protection.

Chitin and chitosan are naturally-occurring compounds that have potential in agriculture with regard to controlling plant diseases. These molecules were shown to display toxicity and inhibit fungal growth and development. They were reported to be active against viruses, bacteria and other pests. Fragments from chitin and chitosan are known to have eliciting activities leading to a variety of defense responses in host plants in response to microbial infections, including the accumulation of phytoalexins, pathogen-related (PR) proteins and proteinase inhibitors, lignin synthesis, and callose formation. Based on these and other proprieties that help strengthen host plant defenses, interest has been growing in using them in agricultural systems to reduce the negative impact of diseases on yield and quality of crops. This review recapitulates the properties and uses of chitin, chitosan, and their derivatives, and will focus on their applications and mechanisms of action during plant-pathogen interactions.

Comparative screening of bacteria for biological control of potato late blight (strain US-8), using in- vitro, detached-leaves, and whole-plant testing systems

Forty-three bacteria were isolated from the phylloplane and (or) rhizosphere of potato and canola plants and tested for their ability to control the pathogen Phytophthora infestans (strain US-8) causing late blight on potato. This study revealed the benefit of using more than one system when searching for biocontrol activity. In this regard, the complementarity of the three systems chosen (in-vitro culture media, detached leaves, and whole plants) in selecting and identifying potential modes of action, provided a useful insight into the different types of biocontrol activity present. Bacteria with biocontrol activity were from the genera Bacillus, Pseudomonas, Rahnella, and Serratia. Mechanisms of inhibition characterized included those occurring directly, through antibiosis, and (or) indirectly, through the induction of plant defense systems.

Biocontrol Treatments Confer Protection Against Verticillium dahliae Infection of Potato by Inducing Antimicrobial Metabolites

Verticillium wilt, caused by Verticillium dahliae Kleb., is a serious potato (Solanum tuberosum L.) disease worldwide, and biocontrol represents a promising eco-friendly strategy to reduce its impact. We used extracts from Canada milk vetch (CMV) and a set of four V. dahliae-antagonistic bacterial strains to coat potato seeds at planting and examined the degree of protection provided against V. dahliae as well as accumulation of soluble phenolics as markers for induced resistance. All tested treatments were effective in reducing disease severity, and CMV showed the highest level of protection. In this treatment, flavonol-glycoside rutin was a highly abundant compound induced in potato tissues, with levels two to three times higher than those detected in noninoculated controls and V. dahliae-inoculated plants. We investigated dose-dependent effects of rutin on V. dahliae growth and sporulation in vitro and in planta. The effect of rutin on mycelial growth was inconsistent between disk assay and amended medium experiments. On the other hand, significant reduction of V. dahliae sporulation in vitro was consistently observed starting at 300 and 100 μM for isolates Vd-9 and Vd-21, respectively. We successfully detected 2-protocatechuoylphloroglucinolcarboxylic acid (2-PCPGCA) using ultra-performance liquid chromatography tandem mass spectrometry, indicating that V. dahliae dioxygenally oxidizes quercetin. Quercetin, as an aglycone, is freed from the sugar moiety by glucosidases and rhamnosidases produced by the fungus and is a substrate for quercetinases. The occurrence of quercetinases in V. dahliae provides a background to formulate a hypothesis about how by-product 2-PCPGCA may be interfering with potato defenses.

Quality evaluation of processing potatoes by near infrared reflectance

The percentage dry matter and reducing sugar content are prime quality parameters for assessing the potential of potato tubers to produce acceptable yields of processed products possessing good textural and colour quality attributes. A study was conducted to develop a near infrared (NIR) calibration for dry matter which would be unaffected by storage time and crop year, and to assess whether NIR could be used to screen tubers with high reducing sugar content from tubers with more acceptable levels. Tubers from commercial growers of French fry processing potatoes were sampled over two years for dry matter and one year for sugars. Tubers used for development of the NIR calibration were stored at 8°C, while those for sugars were stored at 8, 5 and 2°C. A small coin of potato flesh was excised from each tuber, its NIR spectrum obtained on a scanning spectrophotometer and its specific gravity and dry matter, or its reducing sugar content, determined. Correlation plots indicated that wavelengths that correlated well for specific gravity correlated well for dry matter. The standard error of performance for NIR determination of percentage dry matter varied from 1.3 to 1.5%. Calibrations for dry matter were robust and were not affected by time or conditions of storage nor by changeover from one crop year to another. It appears, however, that adjustments to the NIR calibration would be required at harvest time. Coefficients of variation (CV) in determining dry matter by NIR were 5–6%. Spectral averaging appears to have potential for improving on this CV. NIR in reflectance mode was unable to act as a screening tool for unacceptable sugar content in tubers. © 1999 Society of Chemical Industry

Alteration of secondary metabolites' profiles in potato leaves in response to weakly and highly aggressive isolates of Phytophthora infestans

Phytophthora infestans is the cause of late blight, a devastating disease in potato and tomato. Many of the mechanisms underlying P. infestans pathogenesis and defense responses in potato are still unclear. We investigated the effects of P. infestans on the changes in the accumulation of secondary metabolites in potato cultivars using whole plants. Four preformed flavonoids and one terpenoid compound produced in potato tissues were differentially affected by the P. infestans inoculation. In Russet Burbank, the accumulation of catechin and rutin was suppressed by both P. infestans isolates US-11 and US-8, while the flavanone P3 was associated with susceptibility to this pathogen. On the other hand, catechin, flavonol-glycoside P2, and an unidentified terpenoid (T1), may be involved in the defense of cultivar Defender to both tested P. infestans isolates, providing new evidence that different preformed flavonoids and terpenoids in potato may play important roles in its defense or susceptibility to P. infestans. These results add to the pool of data showing the involvement of other phenolics and terpenes in potato resistance to microbial pathogens.

Verticillium dahliae's VdNEP acts both as a plant defence elicitor and a pathogenicity factor in the interaction with Helianthus annuus

Abstract The soil-borne fungus Verticillium dahliae Kleb. causes a serious wilt disease in many economic crops worldwide, including sunflower. We investigated the role of V. dahliaes necrosis and ethylene-inducing proteins (VdNEP) in the interaction with sunflower. We inoculated two highly and two weakly aggressive V. dahliae isolates onto moderately resistant and susceptible sunflower hybrids. We also synthesized VdNEP protein in vitro and infiltrated it into sunflower control plants to compare its effects with those of the fungus. VdNEP induced wilting symptoms, i.e. chlorosis, necrosis and vascular discoloration but also triggered host defence responses, i.e. hypersensitive response in Nicotiana benthamiana leaves and sunflower cotyledons. It activated the production of reactive oxygen species and the accumulation of fluorescent compounds in sunflower leaves, as well as pathogenesis-related genes (Ha-PR-3 and Ha-PR-5), two defensin genes (Ha-PDF and Ha-CUA1) and those encoding Ha-ACO, Ha-CHOX, Ha-GST and Ha-SCO. The latter suggested that more than one signalling pathway may be involved in the V. dahliae-sunflower interaction. Two other genes (Ha-PAL and Ha-NML1), related to the salicylic acid pathway, were slightly downregulated by VdNEP, suggesting a possible involvement of VdNEP in affecting sunflower defences. The phenotypic changes induced by VdNEP indicate that this protein acts both as a defence elicitor and a pathogenicity factor.

Pathogenic variation of Verticillium dahliae after serial passages through potato and sunflower

Verticillium wilt is a limiting factor in many field crops, including potato and sunflower. The causal agent, Verticillium dahliae Kleb., is known for its high level of diversity and pathogenic variability. In an earlier study, we showed such a high variability among isolates recovered from either potato or sunflower when inoculated on their original or alternative hosts. Verticillium dahliae isolates from potato were more aggressive on both potato and sunflower while the ones from sunflower were to a certain extent more adapted to sunflower than to potato. In the present study, we show the ability of weakly aggressive potato isolates to gain pathogenicity on either potato or sunflower when inoculated to a susceptible potato cultivar ‘Kennebec’ for four successive generations. However, some isolates showed a loss in pathogenicity after four successive passages on susceptible potato cultivar ‘Kennebec’, especially on the moderately resistant potato cultivar ‘Ranger Russet’. Results in terms of gain or loss of pathogenicity by potato and sunflower isolates are discussed in relation to the effect of successive passages through their original or alternative hosts. Key words: Verticillium dahliae Kleb., potato, sunflower, host of origin, alternative host, successive passages, pathogenicity gain/loss, pathogenicity index. La verticilliose constitue un facteur limitant pour de nombreuses cultures incluant la pomme de terre et le tournesol. L’agent causal, Verticillium dahliae Kleb., est connu pour son degré élevé de diversité génétique et de variabilité de pouvoir pathogène. Dans une étude précédente, nous avions montré la présence d’une telle variabilité entre des souches isolées soit de pomme de terre soit de tournesol, et inoculées sur leur hôte d’origine ou alternatif. Les isolats de V. dahliae provenant de pomme de terre étaient plus agressifs aussi bien sur pomme de terre que sur tournesol, tandis que ceux provenant de tournesol étaient relativement mieux adaptés sur tournesol que sur pomme de terre. Dans la présente étude, nous montrons la capacité de certains isolats peu agressifs, provenant de pomme de terre, d’acquérir un plus grand pouvoir pathogène, aussi bien sur pomme de terre que sur tournesol, après avoir été propagés pendant quatre générations sur le cultivar sensible de pomme de terre ‘Kennebec’. Par contre, après quatre passages successifs sur le cultivar ‘Kennebec’, certains isolats ont vu leur pouvoir pathogène diminuer, plus particulièrement sur le cultivar partiellement résistant ‘Ranger Russet’. Ces résultats sont discutés en termes de gain ou de perte de pouvoir pathogène, par des isolats provenant de pomme de terre ou de tournesol, dans le but d’élucider l’effet de leur passages successifs sur leurs hôtes d’origine ou alternatif. Mots-clés : Verticillium dahliae Kleb., pomme de terre, tournesol, hôte d’origine, hôte alternatif, passages successifs, gain/perte de pouvoir pathogène, index de pathogenicité.

Vegetative compatibility of Verticillium dahliae isolates from potato and sunflower using nitrate non-utilizing (nit) mutants and PCR-based approaches

Abstract Vegetative compatibility groups (VCGs) of 12 potato and nine sunflower isolates of Verticillium dahliae were determined using both nitrate non-utilizing (nit) mutants and multiplex-nested-PCR approaches. Based on analysis of nit mutants, most potato isolates showed strong compatibility with VCG 4A and weak compatibility with VCG 4B; one potato isolate was also weakly compatible with VCGs 2A, 2B and 3. Sunflower isolates were more variable and most of them showed weak reactions with VCG 4A and 4B. One sunflower isolate, Vs06-14, was compatible only with VCG3 testers, whereas another isolate, Vs06-20, was compatible with all VCG groups except VCG2A. Vegetative compatibility of four selected isolates, two from potato and two from sunflower, was also investigated after passing them through potato or sunflower for four generations. The VCG of one potato isolate did not change, whereas the other one changed from VCG4B to 4A. One sunflower isolate, initially compatible with both VCG4A and 4B, became incompatible with both. The other sunflower isolate was not compatible with any VCG and remained so. In a second part of this work, multiplex-nested-PCR was carried out to characterize VCGs of the same isolates of V. dahliae. Vegetative compatibility grouping using this approach assigned the V. dahliae isolates to VCG1, 2A, 3 or 4A. Six of the 12 potato isolates and four of the nine sunflower isolates of V. dahliae could not be identified or placed in any VCG group based on PCR results. In the present study, the multiplex-nested-PCR results were confirmed using nit mutants. As such, PCR-based methods will require more specific primers before they can replace the approach based on nit mutants. Current address of A.F. El-Bebany: Department of Plant Pathology, Faculty of Agriculture, Alexandria University, El-Shatby, 21545, Alexandria, Egypt.

Plants versus Fungi and Oomycetes: Pathogenesis, Defense and Counter-Defense in the Proteomics Era

Plant-fungi and plant-oomycete interactions have been studied at the proteomic level for many decades. However, it is only in the last few years, with the development of new approaches, combined with bioinformatics data mining tools, gel staining, and analytical instruments, such as 2D-PAGE/nanoflow-LC-MS/MS, that proteomic approaches thrived. They allow screening and analysis, at the sub-cellular level, of peptides and proteins resulting from plants, pathogens, and their interactions. They also highlight post-translational modifications to proteins, e.g., glycosylation, phosphorylation or cleavage. However, many challenges are encountered during in planta studies aimed at stressing details of host defenses and fungal and oomycete pathogenicity determinants during interactions. Dissecting the mechanisms of such host-pathogen systems, including pathogen counter-defenses, will ensure a step ahead towards understanding current outcomes of interactions from a co-evolutionary point of view, and eventually move a step forward in building more durable strategies for management of diseases caused by fungi and oomycetes. Unraveling intricacies of more complex proteomic interactions that involve additional microbes, i.e., PGPRs and symbiotic fungi, which strengthen plant defenses will generate valuable information on how pathosystems actually function in nature, and thereby provide clues to solving disease problems that engender major losses in crops every year.

A cupin domain-containing protein with a quercetinase activity (VdQase) regulates Verticillium dahliae's pathogenicity and contributes to counteracting host defenses.

We previously identified rutin as part of potato root responses to its pathogen Verticillium dahliae. Rutin was directly toxic to the pathogen at doses greater than 160 μM, a threshold below which many V. dahliae pathogenicity-related genes were up-regulated. We identified and characterized a cupin domain-containing protein (VdQase) with a dioxygenase activity and a potential role in V. dahliae-potato interactions. The pathogenicity of VdQase knock-out mutants generated through Agrobacterium tumefasciens-mediated transformation was significantly reduced on susceptible potato cultivar Kennebec compared to wild type isolates. Fluorescence microscopy revealed a higher accumulation of flavonols in the stems of infected potatoes and a higher concentration of rutin in the leaves in response to the VdQase mutants as compared to wild type isolates. This, along with the HPLC characterization of high residual and non-utilized quercetin in presence of the knockout mutants, indicates the involvement of VdQase in the catabolism of quercetin and possibly other flavonols in planta. Quantification of Salicylic and Jasmonic Acids (SA, JA) in response to the mutants vs. wild type isolates revealed involvement of VdQase in the interference with signaling, suggesting a role in pathogenicity. It is hypothesized that the by-product of dioxygenation 2-protocatechuoylphloroglucinolcarboxylic acid, after dissociating into phloroglucinol and protocatechuoyl moieties, becomes a starting point for benzoic acid and SA, thereby interfering with the JA pathway and affecting the interaction outcome. These events may be key factors for V. dahliae in countering potato defenses and becoming notorious in the rhizosphere.