Induced systemic resistance (ISR) is a phenomenon whereby resistance to infectious disease is systemically induced by localized infection or treatment with microbial components or products or by a diverse group of structurally unrelated inorganic or organic compounds.
ISR is most effective against fungi (Kuc, 2000). The yeast cell wall component used was derived from Saccharomyces cerevisiae (Lyons, 1970). The component belongs to a new category of systemic inducers known as plant activators. Preliminary studies indicate it has two modes of action (Newton et al., 1993).
The first is that a thin film of yeast cell wall component is deposited on the leaves and stems of the crop and prevents attachment of the pathogen to the plant tissue. The second, and more important means, is through the development of ISR after it has been taken into the plant.
By application of ISR 2000™ (an extract of the cell wall material of yeast), the expectation is that the crop plant is stimulated into a high state of preparedness and will be better able to defend itself against pathogenic invasion.
There are two conditions that need to be met for this technology to be successful. The first is that systemic resistance is induced well in advance of invasion. The second is that once the systemic resistance has been induced, it must be maintained at a high level during the period that disease prevention is desired. This is normally accomplished by the periodic application of the elicitor. At a biochemical level, peroxidase expression has been shown in several plant systems to be altered by stress chemicals and infection and may be used to indicate activation of ISR (Lagrimini et al., 1993).
The objectives of the work described here were to use ISR 2000™ to improve fungicide efficacy and to obtain better disease control with reduced use of fungicides. Variation in peroxidase activity was determined as a measure of ISR.
Materials and methods
The materials used in both greenhouse and field studies are given in Table 1.
Tomato seedlings were grown in pots under greenhouse conditions (25±2 °C, 8/16 photoperiod).
Table 1. Plant protection products and their rate of use
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To test the elicitation response and the level of disease resistance, plants were treated at the 8-10 leaf stage with ISR 2000™, fungicides and bactericides or just water (control). Treatments were applied three times at seven day intervals.
Pathogens tested were late blight (Phytophthora infestans) and bacterial speck (Pseudomonas syringae pv. tomato). These were applied three days after the first application of ISR 2000™ or chemical, and leaves were collected for peroxidase assay 10 days after the last applications. Fifteen replications were carried out per treatment.
Leaves were harvested, freeze-dried in liquid nitrogen and lyophilized. Crude extracts (0.2 g) were homogenized with 2 ml sodium phosphate buffer (0.05 M, pH 6.5) and centrifuged. The supernatants were collected and their protein concentrations were determined (Bradford, 1976) using bovine serum albumin (BSA) as a standard. The peroxidase enzyme activity was assayed spectrophotometrically (Kanner and Kinsella, 1983).
Two field trials were conducted in 2000 in dripirrigated tomato crops in the research fields of Demko, one of the largest tomato processors in Turkey. Plots contained approximately 250 plants and treatments were replicated four times in a randomized complete block design.
ISR 2000™ was applied twice at 1 litre/ha in 1,000 litres water/ha at 2 and 5 weeks after transplanting. As a grower standard treatment for comparison, copper hydroxide was applied once at seedling stage and copper oxychloride was sprayed at the same time as ISR 2000™.
Plots (3 m) were evaluated for fruit number on 27 June 2000, and harvested on 13 August 2000. The trial field of Demko suffered from a lack of water due to damage to the drip irrigation system for several weeks during the growing season.
There were no quantifiable diseases in the fields possibly because of the very dry conditions.
In 2001, field trials on blight in tomatoes and potatoes were conducted at two locations.
Experiments were of a randomised block design with four replications; each block was 6 rows wide and 10 m long. Products were applied at label recommended rates using a backpack sprayer. First application was made when conditions were conducive for the disease, around first bloom, and continued at 10 day intervals. Treatments were evaluated on 60-75 plants from the middle 4 rows in each plot, using a 0-5 severity scale, when the first fruits reached the harvest stage for tomatoes and 10 days after the last application for potatoes.
Trials on grapevine powdery mildew were carried out using a randomized block design with 5 replicates. Each replicate consisted of 16 vines (4x4). A backpack sprayer was used for thorough spray coverage. Sprays began when the shoots were about 25-30 cm length, in April for Ege region and in May for Marmara region. Second treatments were applied at the time flower petals dropped and berries were forming. Sprays were applied every 2 weeks. Severity of powdery mildew (0-3 scale) was assessed on 100 leaves from the four vine stocks, beginning with the fourth leaf from the bottom.
Peroxidase enzyme activities in tomato leaves were increased greatly after treatment with ISR 2000™.
When treatment was followed by inoculation with Pseudomonas syringae pv. tomato and copper oxychloride, or Phytophthora infestans and ISR 2000TM with Crop-SetTM, there were also large increases in peroxidase activity compared with the control (Table 2).
Table 2. Peroxidase enzyme activity in tomato leaves following treatment with ISR 2000TM and challenge with Phytophthora infestans and Pseudomonas syringae pv. tomato.
*P = 0.05
In greenhouse trials on tomato late blight, the greatest effectiveness was found with ISR 2000TM + fenamidon and mancozeb (80% reduction) (Table 3). ISR 2000TM + copper hydroxide gave the greatest reduction of bacterial speck (Table 4). ISR 2000TM alone was comparably effective against both diseases. These results show that, under greenhouse conditions, alternating a chemical treatment (one spray) with ISR 2000TM (two sprays) was equivalent to the chemical treatment alone (three sprays).
In the tomato field trials in 2000, no foliar disease was observed at either site. Nevertheless, at 7 weeks before harvest, plants treated with ISR 2000TM had significantly more flowers and fruit than the grower standard (Table 5). Under stress conditions related to water shortage, plants treated with ISR 2000TM yielded significantly more tomatoes, and a larger percentage of red marketable fruit, than those treated with the grower standard fungicides (Table 6).
In tomato field tests in 2001, the severity of late blight averaged 37.8% (leaf area affected) and the efficiency of ISR 2000TM, when used in alternation with azoxystrobin, was found to be 87.4%. Azoxystrobin alone showed 87.7% efficacy. There were no statistically significant differences between ISR 2000TM + azoxystrobin treatment and azoxystrobin alone (Table 7). No phytotoxicity was observed following treatment with the yeast extract.
Table 3. Effect of ISR 2000TM and fungicides on tomato late blight (P. infestans).
Table 4. Effect of ISR 2000TM and fungicides on bacterial speck (P. syringae pv. tomato).
Table 5. Effect of ISR 2000TM and fungicides on the number of open flowers and set tomato fruit.
Table 6. Total weight (kg) of tomatoes following treatment with ISR 2000TM or fungicides.
Table 7. Effect of ISR 2000TM and fungicides on tomato late blight (Aegean and Marmara Region), 2001.
ab means differ (P=0.05)
In potato field trials, similar results were obtained.
Alternating ISR 2000TM and azoxystrobin gave 87.4% control, equivalent to that achieved with azoxystrobin alone (Table 8). The use of ISR 2000TM alternating with azoxystrobin proved effective in decreasing the number of fungicide applications necessary when disease severity was medium. ISR 2000TM alone also gave some control of potato blight, although it was not as effective as the other treatments.
In vineyard tests, ISR 2000TM alternating with triadimenol was found to be as effective as triadimenol alone in the control of powdery mildew in grapes (Table 9). ISR 2000TM alone also gave some control.
In summary, the use of ISR 2000 in integrated disease management is environmentally friendly and can help reduce the number of fungicides required to achieve effective control. It may also reduce the cost of disease control and the likelihood of resistance development.
Table 8. Effect of ISR 2000TM and azoxystrobin on potato late blight (Aegean and Marmara Region), 2001.
ab means differ (P=0.05)
Table 9. Effect of ISR 2000TM and triadimenol on grape powdery mildew (Aegean and Marmara Region), 2001.
ab means differ (P=0.05)
We thank TAT and Demko for their help during field tests and Hektas, Aventis, Syngenta and Seres for providing agricultural plant protection materials.
Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.
Kanner, J. and J.E. Kinsella. 1983. Lipid deterioration initiated by phagocytic cells in muscle foods: β-carotene destruction by a myeloperoxidase-hydrogen peroxide-halide system. J. Agric. Food Chem. 31:370-376.
Kuc, J. 2000. Development and future direction of induced systemic resistance in plants. Crop Protection 19:859-861.
Lagrimini, L.M., J. Vaughn, W.A. Erb and S.A. Miller. 1993. Peroxidase over production in tomato: wound-induced polyphenol deposition and disease resistance. Hort. Sci. 28:218-221.
Lyons, T.P. 1970. Biochemistry of the yeast cell wall. Dissertation. University of Birmingham, Birmingham, England.
Newton, A.C., G.D. Lyon and T. Reglinski. 1993. Development of a new crop protection system using yeast extracts. Home Grown Cereal Authority. Project Report No. 78:1-41.