Abstract: Wildfire leaf spot caused by Psuedomonas syringae pv. tabaci + is a important seedbed diseases of Solanaceous crops in Zimbabwe. It results in loses of economic significance in yield quality every season. The objective of this study was to evaluate the efficacy of Acibenzolar-S-methyl (ABM; Actigard 50 WP, Syngenta Inc.) against Wildfire (Pseudomonas syringae pv. tabaci+) in paprika seedlings. ABM was evaluated for its ability to suppress Pseudomonas syringae pv. tabaci tox + of paprika (Capsicum anuum L.) seedlings in the greenhouse. Four ABM spraying treatments differing in concentration and application intervals were evaluated and compared with the standard practice of spraying copper oxychloride at a rate of 1.56 g ai m-2. Paprika seedlings sprayed with ABM were significantly protected from wildfire (Pseudomonas syringae pv. tabaci tox +). The ABM restricted the incidence and severity of wildfire pathogens from progressing within leaf lamina as shown by a lower Area Under Disease Progress Curve (AUDPC) for disease incidence in seedlings sprayed with ABM when compared to seedlings sprayed with copper oxychloride. Since, both levels of the active ingredient confer resistance exhibiting no significant differences in severity and incidence of wildfire, it is therefore more economic to apply lower rates at lower frequencies in the nursery. The ABM may be a useful tool in IPM programs or replacement of pesticide such as copper oxychloride in the management of Pseudomonas syringae pv. tabaci tox +.
INTRODUCTION
Paprika (Capsicum annum) is a high value crop belonging to the Solanaceae family. The food colourant oleoresin and capsaicin used in the cosmetic industry for lipstick and other cosmetics are the major economic products extracted from the condiment paprika. In Zimbabwe it began to be commercially produced in between 1999 and 2000 (Agritex, 2000) and has a promising potential to replace tobacco as a highest export crop, particularly now that the tobacco market is unstable due to anti smoking campaigns globally (Handiseni, 2004). Estimations of world demand for paprika have been pegged between 50 000 and 60 000 tonnes per annum (Agrikor, 2000). Among the major challenges highlighted concerning production have been poor field establishment and a lack of disease management information (Chivinge and Mariga, 2000). Wildfire and angular leaf spot caused by two forms of the same pathogen Psuedomonas syringae pv. tabaci are important seedbed diseases of Solanaceous crops in Zimbabwe and cause losses of economic significance in yield quality every season. The management of the disease is complicated by the occurrence of several races of the pathogen (Cole, 2002). Control by copper based bactericides is currently the most effective method of disease management in the nursery. Copper-based bactericides control the pathogens, but they are used exclusively in the seedbeds because copper residues accumulate (Hartill, 1969) to what are now considered unacceptable concentrations on field leaves. Accumulation of copper residues to unacceptable concentrations and lengthened residual effects of copper bactericides, which increase health, risks by the consumer have raised mounting food safety concerns and pressure from environmentalists and export chemical residual limits to reduce use of pesticides and creating a dilemma for the food processing industry (Bolkan and Ranhert, 1994) this has led to the necessity to opt for alternative control methods.
Plant activators, a category of novel chemicals, induce the defense capabilities of plants. For example, acibenzolar-S-methyl (ABM), a structural analog of salicylic acid. Technological advances in biochemical and molecular biological tools have improved our understanding of plants, pathogens and their interactions, which extensively helped the development of commercial formulations of induced resistance products possible. The ABM is a plant activator that has been reported to suppress fungal, bacterial and viral diseases in several hosts and thus it has been considered a potential tool of crop protection (Louws et al., 2001; Pappu et al., 2000; Riley and Pappu, 2000). The ABM, a benzothiadiazole, is one of a new generation of plant protection products termed activators (Kessmann et al., 1994) or synthetic elicitors (Lyon and Newton, 1997). It protected several tobacco plants against a number of fungal and bacterial diseases (Cole, 2002). Pathogens are less likely to be able to evolve mechanisms to overcome the resistance conferred by Systemic Acquired Resistance (SAR) than they are to overcome resistance of genetically modified plants or to commercial fungicides (Malamy et al., 1996). The ABM can be applied before pathogens infect plants, the SAR it induces is uniformly activated in the whole plant (Cole, 2002). The ABM is one of a new generation of plant protection products termed activators or synthetic elicitors. It has been evaluated against a range of tobacco pathogens in Zimbabwe including wildfire and angular leaf spot in tobacco (Cole, 1999). Wildfire Pseudomonas ssyringae pv. tabaci + produces tab toxin and cause wildfire disease on tobacco. The symptoms appear as necrotic lesions that eventually become surrounded by large chlorotic haloes. The management of the diseases is complicated by the occurrence of several races of the pathogen. Elicitation of SAR and reduced disease incidence or severity has been shown in greenhouse and field experiments on tomato (Solanum lycopersicum L.), bell pepper and long cayenne pepper (Capsicum annuum L.), muskmelon (Cucumis melo L.), watermelon (Citrullus lanatus (Thunb.) Matsum and Nakai var. lanatus), sugar beet (Beta vulgaris L.), tobacco (Nicotiana tabacum L.), Arabidopsis sp., cucumber (Cucumis sativus L.) and loblolly pine (Pinus taeda L.) using Bacillus amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides and B. sphaericus (Kloepper et al., 2004).
The objective of this study was to evaluate the efficacy of acibenzolar-S-methyl against Wildfire (Pseudomonas syringae pv. tabaci+) in paprika seedlings.
MATERIALS AND METHODS
Land preparation and experimental design: The study was carried out in the experimental greenhouse at the Midlands State University in Gweru in 2007. Located 29.8° S and 18, 3° E in the Agro Ecological Zone Natural Region III. Land preparation was done manually using a hoe, it was then harrowed to a fine tilth. Each plot comprised of seedbed measuring 1x12 m. The seedbeds were laid out in a Randomized Complete Block Design with each treatment replicated four times. The seed beds were sterilised by dry heat from burning maize cobs at rate of 8 kg m-2 (Handiseni, 2004). A Compound D (8:14:7 NPK) fertilizer was applied as a basal fertiliser at a rate of 100 g m-2. Paprika seeds were then drilled into 1 cm deep furrows 7 cm apart a week after dry heat sterilization of seedbeds. A thin layer of soil was used to cover the seeds followed by thin sterilised grass mulch which was removed seven days after planting to prevent seedling etiolation after germination. The study was repeated three times.
Inoculum preparation and pathogen inoculation: A week after the first ABM sprays prior to inoculation (7 WAS), the pure cultures of Psuedomonas syringae pv. tabaci + were subjected to serial dilutions in the laboratory. The Psuedomonas syringae pv. tabaci + inoculum used was procured from the Plant Pathology Department at the Tobacco Research Board. The pure culture samples were preserved by looping colonies of the pure cultures onto nutrient agar slants. Aluminum loops were heat sterilised and then used to lift colonies onto agar slants. These samples were incubated at 36.5°C. Some of the culture was loop inoculated onto the nutrient broth in preparation for serial dilutions and stored at 36.5°C. The initial step in the serial dilutions combined one unit volume of pure culture with 9 unit volumes of sterile water to give the initial 100 dilution suspension of bacteria. A dilution series was made from the resulting suspension to obtain a dilution of 107 colony forming units cfu mL-1 by diluting the initial 10 mL suspension into 90 mL of sterile water. Seedlings were inoculated with a suspension of Psuedomonas syringae pv. tabaci + (107 cfu mL-1) one week after the first ABM sprays and immediately after the first copper oxychloride sprays.
Spraying treatments: The ABM spraying treatments were; 0.05 g ai m-1 applied at 6WAS, 7WAS and at 10 days thereafter; 0.05 g ai m-1 applied at 6 WAS, 7 WAS, 8 WAS and at 10 days thereafter; 0.1 g ai m-2 applied at 6WAS and 7 WAS and at 10 days thereafter and 0.1 g ai m-2 at 6 WAS, 7 WAS and 8 WAS at 10 days thereafter. A standard spray of copper oxychloride at 1.56 g ai m-2 applied weekly starting at 7 WAS (farmer practice) and non sprayed control treatments were also included.
Data collection
Disease incidence: Data on disease incidence was obtained by randomly assessing
the presence of chlorotic haloes on 5 randomly chosen plants from a net plot
measuring 50x50 cm. So as to be able to calculate the disease progress on the
plants, the same randomly selected plants were marked and assessed throughout
the study. The number of plants showing symptoms was then expressed as a percentage
of all scored plants. The fomula shown below was used to calculate disease incidence:
Where:
| n | = | No. of plants affected by disease |
| N | = | No. of plants assesd |
Disease severity: The number of haloes was counted on 5 randomly chosen plants within the net plot, number of chlorotic haloes was counted per plant as the symptoms were observed. In addition, the diameter of the observed symptoms of chlorotic haloes were measured. The data was collected at weekly intervals starting at 9 WAS up to 11 WAS.
Data analysis: The data from the three studies were combined after Bartlett’s test for homogeneity showed that the variances were homogenous across the three studies. The disease severity and incidence data were then used to calculate the area under disease progress curves (AUDPC) by the trapezoidal integration program of Sigma Plot 2000 computer package (Shaner and Finney, 1977). The AUDPC data was subjected to analysis of variance to test for significance of the treatment effects using were analyzed with GENSTAT Discovery Edition 1 Release 4.23 (Genstat, 2003). Where F tests were significant (p<0.05), the treatment means were separated using the Least Significant Difference, mean separation technique. AUDPC were calculated before the analysis of variance using the formula (Shaner and Finney, 1977).
Where:
| Yi | = | Disease severity score at time i and |
| Xi | = | Time of scoring (weeks) |
RESULTS
Mean number of haloes per plant: There were significant differences (p<0.05) in the mean number of chlorotic haloes of wildfire per plant when paprika seedlings from plots of different spray treatments were compared to the paprika seedlings from non sprayed control (Table 1). Interestingly the mean number of chlorotic haloes per plant were significantly the same when paprika seedlings sprayed with ABM at different application rates and application intervals when compared to paprika seedlings in plots sprayed with copper oxychloride. Doubling the application of ABM from 0.05 to 0.1 g ai m-2, did not result in a corresponding significant reduction in mean number of haloes per plant.
AUDPC disease incidence: The AUDPC incidence of chlorotic haloes of wildfire was significantly (p<0.05) lower in paprika seedlings from plots sprayed treatments than the paprika seedlings from non sprayed control (Table 1). Seedlings from seedbeds sprayed with copper oxychloride applied at 1.56 g ai m-2 had a significantly higher AUDPC incidence of chlorotic haloes of wildfire when compared to seedlings from seedbeds sprayed with ABM treatments.
Diameter of chlorotic haloes: The diameter of chlorotic haloes in paprika seedlings from unsprayed control seedbeds increased dramatically from 9WAS to 10WAS before stabilising between 10 WAS and 11 WAS (Table 2). The mean diameter of chlorotic haloes in seedlings sprayed with either ABM or copper oxychloride increased from 9 WAS to 10 WAS before it fell at 11 WAS. At 9 WAS and 10 WAS both copper oxychloride spray treatment and ABM conferred the same level of protection with respect to the diameter of chlorotic haloes, which was significantly superior to seedlings from unsprayed control.
| Table 1: | Mean number of chlorotic haloes per plant and AUDPC incidence of wildfire chlorotic haloes on paprika seedlings sprayed with ABM and copper oxychloride |
| †Means within a column having different letters differ significantly (p<0.05) | |
| Table 2: | Diameter of chlorotic haloes on paprika seedlings sprayed with ABM and copper oxychloride |
| †Means within a column having different letters differ significantly (p<0.05) | |
However, at 11 WAS all ABM treatments significantly (p<0.05) reduced mean chlorotic haloes diameter on seedling when compared to seedlings from seedbeds sprayed with copper oxychloride.
DISCUSSION
The efficacy achieved with the use of copper oxychloride with respect to the number of haloes per plant can still be achieved with the use any of the ABM treatments evaluated. Present results are similar to the findings by Cole (2002) who reported that in tobacco seedbeds, although, copper oxychloride significantly reduced the incidence of wildfire, the total resistance of plants treated with ABM was very impressive. Graves and Alexander (2002) also reports that ABM, at a rate of 10.5 g ai ha-1 was equal to or better than the standard copper-based bactericide for controlling bacterial speck and spot, with no adverse affect on yield. This probably can be attributed to that ABM effectively activated plant defenses in such a way that the paprika seedlings were as healthy as seedlings sprayed with copper oxychloride. ABM has no effect on the pathogen, but activates SAR mechanisms (Elmer, 2006). The incidence and severity of wildfire displayed on seedlings suggest that ABM effects on the plants are cumulative rather than instantaneous in conferring a systemic resistance, which is uniform throughout the plants and is long lasting. In tobacco, gene expression associated with SAR induced by Tobacco Mosaic Virus (TMV), involves coordinate accumulation of mRNA from at least nine gene families (Ward et al., 1991) and ABM treatment of tobacco stimulates the same response (Friedrich et al., 1996). This mechanism results in a broad spectrum pathogen resistance which limits the spread of bacterial and fungal pathogens (Cole, 1999) Even though the defense mechanism has been not been studied in paprika, it is likely that ABM mechanism of plant defense is similar to crops already studied.
The superiority of all ABM treatments in reducing the AUDPC disease incidence in paprika seedlings is of fundamental importance in that it implies that ABM treatments will result in seedlings with lesser diseases than those from copper oxychloride treated seedbeds. The ABM has been reported on tomato (Solanum lycopersicum L.), bell pepper and long cayenne pepper (Capsicum annuum L.) disease suppression (Kloepper et al., 2004).
In most cases, healthy seedlings from the seedbed will translate to potentially higher yielding plant. The importance of this is that ABM can achieve this result without any risk or possibility of pestidicide residue on the plants as is the case in the use of copper oxychloride.
The experiment reveals that at a rate of 0.05 g ai m-2, ABM systemic acquired resistance is effectively conferred to seedlings possibly in the same way or to the same level even at twice the amount of ABM per square metre. This result support the general findings reported by Louws et al. (2001) favoring the use of acibenzolar-S-methyl over copper + mancozeb; however, the effective rate of 0.05 g ai ha-1 was used in this study mainly because of the reported phytotoxicity of ABM peppers.
The ABM sprays compared among themselves signified no differences in both severity and incidence. This probably implies that resistance was effectively conferred, regardless of the number of sprays. Cole (1999) reported that seedlings sprayed with ABM had no symptoms of wildfire, even after inoculation with the pathogen and this would ensure that the farmer had sufficient disease-free seedlings for the field crop without the expense and inconvenience of obtaining seedlings from another source.
From these studies, ABM consistently limited the wildfire pathogen from spread and hence effectively reduced disease incidence and severity in paprika seedlings regardless of application rates and different application interval.
The timing of defence responses is critical and can be the difference between being able to cope, or succumbing to the challenge of a pathogen. The ABM has the advantage that it can be applied before any anticipated crop disease. Since, both levels of the active ingredient confer resistance exhibiting no significant differences in severity and incidence of wildfire, it is therefore more economic to apply lower rates at lower frequencies in the nursery. Cost of ASM application is fairly expensive (roughly $ 2.00USD g-1 Actigard 50 WP), but when applied to low acreage seedling nurseries, the cost may be practical.
Present findings confirms the efficacy of ABM in suppression of plant diseases reported in other crops (Cole, 1999, 2002; Friedrich et al., 1996; Elmer, 2006) and hence its potential to be used in integrated pest management systems.
A spraying application ABM at a rate of 0.05 g m-2, at 6, 7 WAS and 10 days thereafter, is therefore recommended in paprika seedling production.
ACKNOWLEDGMENTS
Authors would like to thank the Midlands State University Research Board for providing funds for the research project and the Tobacco Research Board Department of Plant pathology for providing the Psuedomonas syringae pv. tabaci + isolate.