ASM-treated Plants Research Articles

Transient Changes in Defence Gene Expression and Phytohormone Content Induced by Acibenzolar-S-Methyl in Glasshouse and Orchard Grown Kiwifruit

Acibenzolar-S-methyl (ASM), the active ingredient in Actigard® (Syngenta), is a plant defence elicitor used for the management of bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) in kiwifruit. We examined changes in gene expression and phytohormone content in two kiwifruit cultivars, ‘Hayward’ (Actinidia chinensis var. deliciosa) and ‘Zesy002’ (A. chinensis var. chinensis), following ASM application. In glasshouse studies, ASM-induced resistance to stem inoculation with Psa was accompanied by upregulation of salicylic acid (SA) defence pathway genes (PR1, PR2, and PR5) in stems and leaves. The expression of PR1 in ASM-treated plants increased by c. 5-fold in ‘Hayward’ and by over 15-fold in ‘Zesy002,’ relative to the untreated controls, whereas PR2 and PR5 increased by up to 4-fold in both cultivars. Orchard studies over three seasons confirmed the utility of PR1, PR2, and PR5 for monitoring ASM-induced responses in mature vines. PR1 and PR5 were more strongly induced by ASM than PR2 and gene upregulation in ‘Hayward,’ was accompanied by a reduction in Psa leaf spotting; no such relationship was measurable for ‘Zesy002’ because leaf spot symptoms are rarely expressed. In the third season, six additional gene candidates, BAD, Gluc2, Class IV Chit, EDS1A, NPR3, and NIMIN2, were responsive to ASM in ‘Hayward’ and ‘Zesy002.’ Gene upregulation was transient, with expression levels increasing by 1 d after ASM application and declining to control levels between 7 and 14 days. Moreover, the amplitude of gene upregulation depended on leaf developmental stage and was greater in the first true leaf and the youngest mature leaves than in immature leaves along the same shoot. Phytohormone content did not show a repeatable response pattern to ASM in potted plants or in vines possibly as a consequence of their wider role in regulating plant growth and mediating environmental responses. In conclusion, this study demonstrates that defence gene expression can be used to monitor responsiveness to ASM in two genetically distinct mature kiwifruit cultivars (‘Hayward’ and ‘Zesy002’) under orchard conditions. The use of defence marker genes could be of broader utility across kiwifruit species and could be used to guide ASM application schedules in the orchard.

Actinidia-Pseudomonas syringae pv. actinidiae interaction: differentially expressed plant transcripts during infection

Since 2008, kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) has spread to pandemic scale. In order to elucidate the molecular events of the kiwifruit-Psa interaction, Illumina RNAseq was conducted after Psa inoculation and mock inoculation, on both acibenzolar-S-methyl (ASM) treated and untreated Actinidia chinensis plants. De-novo assembly of the kiwifruit transcriptome revealed 39,607 transcripts. To investigate the transcriptome expression profile during the infection process, hierarchical cluster analysis was performed. During the infection process, about 3000 transcripts were modulated. It was notable that the number of modulated transcripts in infected plants were higher than in ASM-treated infected plants. Furthermore, the RNAseq technology permitted us to identify novel transcripts of unknown function but related to infection and ASM treatment. The analysis revealed that the coordinates of some important gene functional categories change, such as hormonal balance, transcriptional regulation and signalling. Different transcription factors were also linked with signalling and hormonal balance and are involved in the ability of ASM-treated plants to assemble the right defence response against Psa. On the other hand, control plants respond to Psa, but not in an adequate way to counteract the infection. Indeed, several transcripts related to plant signalling and putatively encoding RLK-LRR and PRR proteins were exclusively found under these conditions, while they were absent in ASM-treated plants, highlighting how the response of susceptible plants belongs to a separate cluster from that of ASM-treated plants.

Evaluation of acibenzolar-S-methyl-induced resistance against iris yellow spot tospovirus

Acibenzolar-S-Methyl (ASM) is a functional analog of salicylic acid (SA) that activates local and systemic acquired resistance (SAR) responses in plants against a wide variety of pathogens. Iris yellow spot virus (IYSV) is an economically important tospovirus of onion that causes severe economic losses to both bulb and seed crops. IYSV resistant onion cultivars are not available to date and there are limited control options. To explore the possibility of utilizing SAR as a control option, we first used two different hosts of IYSV, Datura stramonium and Nicotiana benthamiana, to study the ability of ASM in triggering SAR against IYSV infection. Quantitative descriptors based on both symptom expression and relative levels of IYSV nucleoprotein and viral small RNA were developed and used to determine the SAR in ASM- and buffer-treated plants. A significant reduction in virus levels in ASM-treated plants was noticed by ELISA and PCR. The level of SAR response was also assessed by measuring the IYSV lesion size and number on the inoculated leaves of ASM-treated plants. ASM-treated plants showed reduced symptoms compared to buffer-treated plants. This study could be useful in potentially developing novel SAR-based options for virus management.

Fontes de fosfito e acibenzolar-S-metílico associados a fungicidas para o controle de doenças foliares na cultura da soja

The goal of this study was to evaluate commercial sources of phosphite and acibenzolar-S-methyl (ASM) followed by one fungicide application on the control of downy mildew, powdery mildew and asian soybean rust, reducing the number of fungicide applications and increasing the control spectrum of these soybean diseases. Two field experiments were carried out in Ponta Grossa, Parana state, Brazil, during the 2006/07 and 2007/08 growing seasons. The experimental design was of randomized blocks with four replications. Treatments consisted on five sources of potassium phosphite and one of manganese (450 g P2O5 ha-1) and ASM (12.5 g a.i. ha-1) sprayed at the V7 and R2 stages. Phosphite- and ASM-treated plants received one application of the fungicide piraclostrobine + epoxiconazol (66.5 + 25 g i.a. ha-1) at the R3 stage and were compared to plants which received two fungicide applications at the R2 and R5.1 stages, as well as to untreated plants. The use of phosphite and ASM significantly reduced the AUDPC for downy mildew. No effects were observed for powdery mildew and Asian rust. The higher number of fungicide application increased the control of biotrophic fungi under higher disease pressure.

Transcriptome profile of acibenzolar-S-methyl-induced genes in tomato suggests a complex polygenic effect on resistance to Phytophthora infestans

Induced resistance by chemicals such as acibenzolar-S-methyl -ASM (commercialized as Actigard by Syngenta Inc) mimics the biological activation of systemic acquired resistance (SAR). ASM takes the place of salicylic acid (SA) in the SAR signal pathway inducing the same molecular markers and range of resistance. The goal of our work was to understand the downstream molecular events by which ASM confers resistance to Phytophthora infestans in tomatoes. To accomplish this goal we assayed gene expression in ASM-treated plants using a microarray with more than 12,000 tomato ESTs. As many as 300 genes were responsive to ASM. Of these, 117 were detected in most of the biological replications. Basal defense associated genes as well as SAR and disease resistance genes (R-like) involved in induced resistance and effector-triggered immunity were highly expressed. We attempted to determine the phenotype of 13 of these genes by virus induced gene silencing (VIGS). These 13 genes were selected on the basis of previous implication in plant defense response and by reliability of induction by ASM. VIGS was partially successful for three of the 13 genes, but this partial silencing did not lead to a significant reduction in the effect of ASM. The ethylene pathway was also activated in response to ASM, but a tomato mutant not responsive to ethylene remained responsive to ASM. It seems most likely that the ASM effect is complex and polygenic, depending on the effect of several genes.

Parents lend a helping hand to their offspring in plant defence

Plants under attack by pathogens and pests can mount a range of inducible defences, encompassing both chemical and structural changes. Although few reports exist, it appears that plants responding to pathogen or herbivore attack, or chemical defence elicitors, may produce progeny that are better able to defend themselves against attack, compared with progeny from unthreatened or untreated plants. To date, all research on transgenerational effects of biotic stress has been conducted on dicotyledenous plants. We examined the possibility that resistance induced by application of chemical defence elicitors to the monocot plant barley, could be passed on to the progeny. Plants were treated with acibenzolar-S-methyl (ASM) or saccharin, and grain harvested at maturity. Germination was unaffected in seed collected from plants treated with saccharin, while germination was reduced significantly in seed collected from ASM-treated plants. The subsequent growth of the seedlings was not significantly different in any of the treatments. However, plants from parents treated with both ASM or saccharin exhibited significantly enhanced resistance to infection by Rhynchosporium commune, despite not being treated with elicitor themselves. These data hint at the possibility of producing disease-resistant plants by exposing parent plants to chemical elicitors.

Accumulation of H2O2 in xylem fluids of cucumber stems during ASM-induced systemic acquired resistance (SAR) involves increased LOX activity and transient accumulation of shikimic acid

Systemic acquired resistance (SAR) to Colletotrichum orbiculare was induced in young cucumber (Cucumis sativus) plants within 3 h of ASM (acibenzolar-S-methyl) application onto the first leaves. A potent signal associated with significant accumulation of hydrogen peroxide in xylem fluids from severed stems appeared to be rapidly translocated from elicited lower leaves within 3 h and 6 h after treatment. Some metabolites of the shikimate, phenylpropanoid and lignin biosynthetic pathways were quantified and significant increases in the levels of shikimic acid were observed in ASM-treated plants challenge-inoculated with the anthracnose fungus. Furthermore, the expression of the 5-enolpyruvylshikimate-3-phosphate synthase gene (EPSPS) was 1.5 times higher within 12 h after ASM treatment in challenge-inoculated plants than in the untreated control. The involvement of lipoxygenase activity, shikimic acid and others such as caffeic acid in the induction of SAR is discussed.

Activities of antioxidant enzymes and photosynthetic responses in tomato pre-treated by plant activators and inoculated by Xanthomonas vesicatoria

The activities of antioxidant enzymes and photosynthetic responses were investigated in tomato ( Lycopersicon esculentum L. var.) pre-treated by plant activators and inoculated by Xanthomonas vesicatoria. Plants were sprayed with acibenzolar-S-methyl, ASM [Bion ® 50 WG (0.2 g l −1)] and aqueous extract from dry necrotic tissue flour (VLA) of ‘Lobeira’ ( Solanum lycocarpum) bush. Four days later, the plants were challenged with a virulent strain of X. vesicatoria, under greenhouse conditions. Tomato leaves were then assessed to determine the activities of the main antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX). A reduction of bacterial leaf spot severity was observed in plants treated by ASM (49.3%) and VLA (31.4%), without any in vitro inhibitory activity over the pathogen. Controls showed decreases in CO 2 assimilation, transpiration, photosynthetic rates, and stomatal conductance. Water use efficiency and carboxylation efficiency were strongly affected in ASM- and VLA-treated, in comparison to controls and healthy plants. The tested substances induced increases in SOD and CAT activities in a delayed enzymatic response typical in compatible plant–pathogen interactions. Measured at daily intervals, activities of APX and POX were significantly ( ρ ⩽ 0.05 ) higher in treated plants than in controls, except for APX in ASM-treated plants where no difference was found when compared to controls. Only POX was clearly induced at the earlier stages after spraying the tomato plants with ASM or VLA. Our results suggest that late increases in antioxidant enzyme activities may play a role in mitigating oxidative damage in restoring the photosynthetic imbalance imposed by the expansion of bacterial lesions.

Effects of acibenzolar- S-methyl on the suppression of Fusarium wilt of cyclamen

Acibenzolar- S-methyl (ASM; Actigard 50 WP ®, Syngenta Inc.) was evaluated for its ability to suppress Fusarium wilt of cyclamen in the greenhouse. The effect of applying increasing rates of ASM to cyclamen seedlings grown in potting mix infested with Fusarium oxysporum f. sp. cyclaminis resulted in a negative relationship between estimates of disease progress values (area-under-the-disease-progress-curve, AUDPC) and ASM rate. Disease symptoms were still evident in most plants, but some ASM-treated plants remained asymptomatic for the entire period. Dry mass of plants grown in infested potting mix were proportionally increased as ASM concentrations increased. In the absence of the pathogen, however, increasing rates of ASM resulted in linear reductions in dry mass. Flower number and quality were not affected by ASM rate. When seedlings were sprayed with 50 μg a.i. ASM ml −1 and then grown in potting mix infested with increasing densities of F. oxysporum f. sp . cyclaminis, disease progress was significantly less than untreated seedlings, but there was variation among repetitions of the experiment. Dry mass was similarly greater than untreated seedlings. The major benefit of ASM was that it delayed the onset of wilt symptoms in most plants for up to 3 weeks and kept a few plants completely asymptomatic. Although applications resulted in no visible phytotoxicity on the leaves, the decline in dry mass may suggest some level of inhibition due to ASM. ASM may still be a useful component of an integrated disease management program for Fusarium wilt of cyclamen.

Acibenzolar-S-methyl induced resistance to Phytophthora capsici in pepper leaves

The leaves of pepper (Capsicum anuum L.) were inoculated with Phytophthora capsici Leonian 3 d after treatment with acibenzolar-S-methylbenzo [1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester (ASM) and resistance to Phytophthora blight disease was investigated. Results showed that P. capsici was significantly inhibited by ASM treatment by up to 45 % in planta. The pepper plants responded to ASM treatments by rapid and transient induction of L-phenylalanine ammonia-lyase (PAL), increase in total phenol content and activities of chitinase and β-1,3-glucanase. No significant increases in enzyme activities were observed in water-treated control plants compared with the ASM-treated plants. Therefore it may be suggested that ASM induces defense-related enzymes, PAL activity, PR proteins and phenol accumulation in ASM-treated plants and contribute to enhance resistance against P. capsici.

Systemic Acquired Resistance Delays Race Shifts to Major Resistance Genes in Bell Pepper

ABSTRACT The lack of durability of host plant disease resistance is a major problem in disease control. Genotype-specific resistance that involves major resistance (R) genes is especially prone to failure. The compatible (i.e., disease) host-pathogen interaction with systemic acquired resistance (SAR) has been studied extensively, but the incompatible (i.e., resistant) interaction less so. Using the pepper-bacterial spot (causal agent, Xanthomonas axonopodis pv. vesicatoria) pathosystem, we examined the effect of SAR in reducing the occurrence of race-change mutants that defeat R genes in laboratory, greenhouse, and field experiments. Pepper plants carrying one or more R genes were sprayed with the plant defense activator acibenzolar-S-methyl (ASM) and challenged with incompatible strains of the pathogen. In the greenhouse, disease lesions first were observed 3 weeks after inoculation. ASM-treated plants carrying a major R gene had significantly fewer lesions caused by both the incompatible (i.e., hypersensitive) and compatible (i.e., disease) responses than occurred on nonsprayed plants. Bacteria isolated from the disease lesions were confirmed to be race-change mutants. In field experiments, there was a delay in the detection of race-change mutants and a reduction in disease severity. Decreased disease severity was associated with a reduction in the number of race-change mutants and the suppression of disease caused by the race-change mutants. This suggests a possible mechanism related to a decrease in the pathogen population size, which subsequently reduces the number of race-change mutants for the selection pressure of R genes. Thus, inducers of SAR are potentially useful for increasing the durability of genotype-specific resistance conferred by major R genes.

INDUCTION OF DISEASE RESISTANCE AND ANTIOXIDANT ENZYMES BY ACIBENZOLAR-S-METHYL AGAINST BACTERIAL CANKER (CLAVIBACTER MICHIGANENSIS SUBSP. MICHIGANENSIS) IN TOMATO

SUMMARY The plant defence activator acibenzolar-S-methyl (Bion) was assayed on tomato seedlings for its ability to induce multicomponent defence response against Clavibacter michiganensis subsp. michiganensis (Cmm). Pretreatment of plants with ASM reduced the severity of the disease as well as the growth of the bacteria in planta. In ASM-treated plants, reduction in disease severity (up to 75%) was correlated with the suppression of bacterial growth (up to 68.2%) during the time course of infection. In plants treated with ASM, activities of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione S-transferase (GST) were estimated as markers of resistance. The results indicated that ASM treatment led to enhanced activities of SOD and GST in tomato leaves. A slightly increase in SOD and GST activities was also found in Cmm-infected leaves. However, the increase in enzyme activities occurred much more rapidly and was more strongly enhanced in Cmminfected leaves that were previously treated with ASM. The involvement of both enzymes in ASM mediated resistance in tomato is discussed.

Induction of disease resistance by the plant activator, acibenzolar- S-methyl (ASM), against bacterial canker ( Clavibacter michiganensis subsp. michiganensis) in tomato seedlings

The plant defence activator acibenzolar- S-methyl (benzo [1,2,3]thiadiazole-7-carbothioic acid- S-methyl ester, ASM; Bion 50 WG) was assayed on tomato seedlings for its ability to induce resistance against Clavibacter michiganensis subsp. michiganensis ( Cmm), the causal agent of bacterial canker of tomato. Pre-treatment of plants with ASM reduced the severity of the disease as well as the growth of the bacteria in planta. In ASM-treated plants, reduction in disease severity (up to 75%) was correlated with suppression of bacterial growth (up to 68.2%) during the time course of infection. In plants treated with ASM, activities of peroxidase (POX) and glutathione peroxidase (GPX) were determined as markers of resistance. Applications of ASM induced a progressive and significant increase of both enzymes in locally treated tissues. Such responses were expressed earlier and with a much higher magnitude when ASM-treated seedlings were challenged with the pathogen, thus providing support to the concept that a signal produced by the pathogen is essential for triggering enhanced synthesis and accumulation of these enzymes. No such activities were observed in water-treated control plants. Therefore, the slower symptom development and reduction in bacterial growth in ASM-treated plants might be due to the increase in activity of both oxidative and antioxidative protection systems in planta.

Acibenzolar- S -methyl-induced resistance to sunflower rust (Puccinia helianthi) is associated with an enhancement of coumarins on foliar surface

Exogenous applications of acibenzolar- S -methyl (ASM) induced resistance to rust infection in sunflower, characterized by reduced infection frequency with no effect on latency period or pustule size, and no increase in host cell necrosis. Cytological studies showed that the reduced frequency of infection was due to a reduction in germination and appressorium formation, while stoma penetration, growth of infection hyphae and haustorium formation remained unaffected. Germination and germtube growth were not hampered by the direct application of ASM on urediospores. The data suggested that ASM had an effect on the production and secretion of fungitoxic compounds to the leaf surface that hamper rust urediospore germination and appressorium formation. This hypothesis is supported by the following experimental results: (i) an increase in the amount of accumulated and excreted coumarins and other phenolic compounds in ASM-treated plants, and (ii) a reduction of germination and of appressorium formation when ayapin, scopoletin, and leaf exudates collected from ASM-treated plants were applied exogenously.