EVALUATION OF ESSENTIAL OILS AS SEED TREATMENT ... - SIPaV

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Journal of Plant Pathology (2012), 94 (2), 273-281

Edizioni ETS Pisa, 2012

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EVALUATION OF ESSENTIAL OILS AS SEED TREATMENT FOR THE CONTROL OF XANTHOMONAS spp. ASSOCIATED WITH THE BACTERIAL LEAF SPOT OF TOMATO IN TANZANIA E.R. Mbega1,2, R.B. Mabagala2, C.N. Mortensen1 and E.G. Wulff1 1Danish

Seed Health Centre for Developing Countries, Department of Agriculture and Ecology, University of Copenhagen, Hoejbakkegaard, Allé 13, 2630 Taastrup, Denmark 2African Seed Health Centre, Department of Crop Science and Production, P.O.BOX 3005, Sokoine University of Agriculture, Morogoro, Tanzania

SUMMARY

Bacterial leaf spot (BLS) caused by Xanthomonas spp. is a serious and a major constraint to tomato production worldwide. The seed-borne nature of BLS, the complex nature of the disease, which is caused by different bacterial species and the current ineffective control measures have made the evaluation of alternative control compounds for seed treatment necessary. Therefore, the efficacy of 11 essential oils applied as seed treatments to control Xanthomonas perforans in tomato and the effect on seed germination and seedling growth was evaluated. Seed treatment with oils of eucalyptus (Eucalyptus globules Labill.), rosemary (Rosmarinus officinalis L.) and niaouli (Melaleuca viridiflora Sol. ex Gaertn.) at 2% concentration inhibited the growth of X. perforans in the in vitro assays and consistently reduced the incidence and severity of BLS in planta tests. Negative effects on seed germination and seedling growth were not observed when tomato seeds were treated with the essential oils of eucalyptus and rosemary. Therefore, eucalyptus and rosemary oils can be used for seed treatment to control BLS in tomato. Further investigation is required on the shelf life of these oils, mode of action and their effects on other seedborne pathogens of tomato and in other crop systems in Tanzania. Key words: Bacterial disease, xanthomonads, control, essential oils, seed treatment.

INTRODUCTION

Bacterial leaf spot (BLS) caused by Xanthomonas spp. is a serious disease and a major constraint to tomato production worldwide. Infected plants show different types of leaf spot and fruit symptoms, yield reduction and poor fruit quality (Kaaya et al., 2003; Abd-Alla

Corresponding author: E.R. Mbega Fax: +255.232.600574 E-mail: [email protected]

and Bashandy, 2008). Tomato is an important cash crop and constitutes 39% of the total vegetable consumption in Tanzania, providing vitamins and minerals to the diet of the population (Tarimo and Sibaway, 1998; Kashina et al., 2002). Although there is a high demand of tomato for fresh market and canning, the national average yield remains low (Non-Womdim et al., 1996). Factors contributing to the low yield include insect pests and diseases; bacterial diseases accounting for 45% of the yield losses (Kaaya et al., 2003). Xanthomonas euvesicatoria, X. vesicatoria, X. perforans and X. gardneri have been reported as the main causal agents of BLS (Jones et al., 2004), but also other bacteria, such as X. campestris pv. raphani (Punina et al., 2009) and X. arboricola (Myung et al., 2010), can be pathogenic to tomato. The BLScausing pathogens survive in seeds, plant debris and volunteer plants (Kaaya et al., 2003). Management of BLS is difficult (Marco and Stall, 1983) and the efficacy of current management practices is limited. Sanitary conditions such as keeping the field weed-free have been recommended and correlate positively with low BLS incidence and severity (Shenge, 2006; Shenge et al., 2010). The cultivation of popular tomato cultivars of high market value such as Moneymaker has been abandoned by producers in Tanzania due to their susceptibility to BLS (Shenge, 2006). Survey conducted in the country revealed that BLS disease of tomato is widespread, affecting all grown cultivars (Black et al., 2001; Shenge et al., 2010). Unfortunately, no resistant tomato cultivars are available for small farm holders and commercial producers in Tanzania (Shenge et al., 2007b). Copper-based fungicides provide limited control in the form of reduction of disease severity. However, resistance to copper used in the control of BLS has been reported in the country (Shenge, 2006). The large number of races reported in tomato and pepper and the different species causing BLS (Jones et al., 2004; Shenge et al., 2007a) has made the control of the disease even more difficult. Antibacterial compounds of plant origin have been widely applied in the cosmetic and food industry and in medicine (Manou et al., 1998; Hammer et al., 1999; Christoph and Stahl-Biskup, 2001; Pradhanang et al., 2003). In agriculture, pesticides of plant origin have


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Control of Xanthomonas spp. in tomato with essential oils

been used for controlling fungal and bacterial infections in a number of crops (Prakash and Roa, 1997; Mohana and Raveesha, 2006). Iacobellis et al. (2005) reported on the efficacy of essential oils from Cuminum cyminum and Carum carvi in the control of several plant bacterial pathogens. Likewise, Mohana and Raveesha (2006) evaluated the efficacy of several oils of plant origin against bacterial diseases, including the BLS pathogen of tomato in in vitro assays, revealing anti-bacterial potential. Essential oils such as thymol, eucalyptus, palmarose and others have also shown antibacterial activity (Ji et al., 2005; Upadhyay et al., 2010). The nature of the BLS diseases and the current ineffective control measures has made relevant the evaluation of new, alternative control practices, such as the use of essential oils as seed treatment of tomato. In this study, we evaluated the efficacy of 11 essential oils of plant origin to control bacterial leaf spot xanthomonads of tomato in seed treatments and their effect on seed germination and seedling growth of tomato. Tomato seed was targeted because it is considered to be one of the main sources of inoculum for BLS-causing pathogens (Kaaya et al., 2003).

MATERIALS AND METHODS

Tomato seed samples. Seeds of tomato (Solanum ly-


copersicum Mill.), cv. Tanya, Cal-J, Roma VF and Moneymaker [tested by dilution assays (Kaaya et al., 2003) and found free from Xanthomonas spp. infection] were used in the experiments. The seeds were surface disinfected in 70% ethanol for 1 min followed by dipping in 1% sodium hypochlorite for 3 min and rinsing three times in sterile distilled water (SDW). After disinfection, the seeds were transferred to Petri dishes containing sterile filter papers, allowed to air-dry overnight in a horizontal air flow clean bench and stored at 4°C until used. Seed inoculation with bacterial suspensions. Surface-disinfected seeds of cv. Tanya were inoculated with Xanthomonas perforans strain NCPPB 4321. This strain was selected amongst other BLS-causing xanthomonads such as X. euvesicatoria strain NCPPB 2968, X. vesicatoria strain NCPPB 422 and X. gardneri strain NCPPB 881 (Jones et al., 2004) due to its ability to cause severe BLS symptoms on tomato cultivars grown in Tanzania. Inoculum suspensions were prepared from cultures of X. perforans that were grown on nutrient agar (meat extract 3 g, bacto peptone 5 g, bacto agar 20 g, distilled water 1000 ml) at 28°C for 24 h. Bacterial cell suspensions were obtained from plate washed with 10 ml of SDW. The suspensions were homogenized by vortexing and adjusted with SDW to a concentration of ca.108

Table 1. The antibacterial activity of eleven essential oils used in seed treatment of tomato cv. Tanya in in vitro (CFU/ml) and in planta assays.

Treatment Controls Sterile distilled water Copper sulphate Untreated seed Essential oils Rosemary Niaouli Eucalyptus Clove Thyme Lemon grass Bergamot Ylang-Ylang Cedar Cypress Frankincense Mean F test

In vitro No. of CFU/ml on NA

No. of plants emerged/ No. of seed sown

Disease incidence d) (%)

In planta Disease severity index e)

>1.00 x 107 0.00 N/A

16/16 16/16 16/16

93.75 0.00 0.00

3.89a 1.00c 1.00c

12.75b 15.88a 16.01 a

0.43b 0.72a 0.74a

1.54c 1.91ab 1.92ab

0.00 0.00 0.00 0.00 0.00 0.00 4.20 x 103 >1.00 x 107 >1.00 x 107 >1.00 x 107 >1.00 x 107 ND

16/16 16/16 16/16 0/16 0/16 0/16 16/16 16/16 16/16 16/16 16/16 ND

0.00 0.00 0.00 N/A N/A N/A 18.75 12.50 25.00 31.25 62.50 ND

1.00c 1.00c 1.00c N/A N/A N/A 1.48c 1.18c 1.53c 2.03c 3.08b 1.67 ***

16.19a 15.28a 16.00a N/A N/A N/A ND ND ND ND ND 15.35 ***

0.74a 0.58a 0.70a N/A N/A N/A ND ND ND ND ND 0.65 ***

1.98a 1.76b 1.98a N/A N/A N/A ND ND ND ND ND 1.85 ***

Height (cm) Fresh weight Width (mm) (g)

d) Disease incidence = number of seedling with bacterial leaf spot symptoms in percentage. e) Disease severity index based on Horsfall and Barrett (1945) scale of 1-6 with modification, where 1= no disease and 6 = >50% BLS symptoms on leaves. Mean followed by same letters in a column are not significantly different at P ≤ 0.05. *** = significant at 1%. N/A = not applicable. No. = abbreviation for ‘number’. ND = not done.


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CFU/ml (OD600 = 0.01). Thereafter, 1,000 tomato seeds were vacuum infiltrated for 30 min with 10 ml of bacterial suspension, then were allowed to air-dry under a horizontal air flow clean bench and kept at 4°C until use, within 1-3 months from inoculation. Seed treatment with essential oils. Essential oils from 11 different plant species (Table 1) obtained from Natur-Drogeriet A/S (Denmark) were used in this study. Twenty pre-inoculated seeds were treated with 1 ml of 2% essential oil diluted in SDW in Eppendorf tubes and placed on agitation table at 100 rpm overnight at 25°C. This essential oil concentration (2%) was selected as optimum based on preliminary testing results of different oil concentrations of 0.5, 1, 2 and 4% with pre-inoculated tomato seeds. Treatments with copper sulphate (200 µg/ml CuSO4.5H2O) and SDW served as controls. After treatment, seeds were blotdried and allowed to air-dry under a horizontal air flow clean bench for 1 h. In vitro evaluation of antibacterial activity. Washings (100 µl) from treated seeds were collected using sterile pipettes and samples were serially diluted to 10-2 with SDW; aliquots of 50 µl from each washing dilution were spread onto nutrient agar (NA) using a sterile glass rod. Colony counts were made 96 h after incubation at 28°C. Colonies were presumed to be X. perforans based on morphology and colour compared to the pure culture and to the colonies found in the plates inoculated with seed washings. The identity of suspected colonies from each plate was confirmed by pathogenicity tests that were conducted as described below. In planta evaluation of antibacterial activity. Tomato seeds treated as described earlier were sown in pots of 8 cm in diameter containing peat soil (Pindstrup substrate No. 2, Pindstrup Mosebrug, Denmark) and sand (3:1), respectively, and kept in a growth chamber at 25-30°C

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under >85% relative humidity (RH). Twenty-one days after sowing, BLS disease incidence and severity were evaluated. Disease incidence was assessed by counting the number of seedling with BLS symptoms and dividing it by the total number of emerged seedlings per treatment. Disease severity was determined based on the Horsfall and Barrett (1945) scale of 1- 6 with modifications (Shenge, 2006), where 1 = no disease, 2 = >03% of the leaves showing BLS symptoms, 3 = >3-12% of the leaves showing BLS symptoms, 4 = >12-25% of the leaves showing BLS symptoms, 5 = >25–50% of the leaves showing BLS symptoms and 6 = >50% of the leaves showing BLS symptoms. The evaluations were repeated in three independent experiments for the essential oils that showed the best ability to reduce BLS in the initial screening tests. Effect on seed germination, seedling vigour and plant dry weight. In order to evaluate the effect of selected essential oils on seed germination, seedling vigour and weight, 1,000 seeds of BLS-free tomato cv. Tanya were soaked in 10 ml of 2% essential oil in vials 9.5 x 2.6 cm in size and placed on agitation table at 100 rpm overnight at 25°C. The seeds were then transferred to Petri dishes containing sterile filter papers and left to air-dry overnight in a horizontal air flow clean bench. Seed germination tests were conducted using the standard International Seed Testing Association (ISTA) top of paper method (ISTA, 2005) with samples of 400 tomato seeds per treatment. Eight replications of 50 seeds were plated uniformly onto three layers of moist blotter paper in a plastic container, kept at 25-28ºC and RH >85% for 14 days. Evaluation of germination was done in accordance with the International Rules for Seed Testing by counting the number of normal (showing potential for development into satisfactory plant under favourable conditions) and abnormal (not showing potential for development into satisfactory plant under favourable conditions) seedlings and dead seeds (ISTA,

Table 2. The effect of rosemary, niaouli and eucalyptus essential oils used as seed treatment on the growth of tomato seedlings of cv. Tanya. Treatment Controls Sterile distilled water Copper sulphate Untreated seed Oil source Rosemary Niaouli Eucalyptus Mean F test

Root length (cm)

Seedling growth parameters Shoot length (cm) Vigour index

Dry weight (g)

3.12a 3.21a 3.43a

3.68a 3.73a 3.64a

531.91a 562.24a 565.80a

0.14a 0.14a 0.14a

3.64a 3.70a 3.78a 3.48 ns

3.94a 5.68a 3.63a 3.70 ns

615.67a 555.97a 555.94a 564.59 ns

0.14a 0.14a 0.14a 0.14 ns

Means followed by same letters in a column are not significantly different based on SNK at P ≤ 0.05. ns = not significant


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2005). Vigour test involved evaluation of root and shoot length of seedlings and the percentage of seed germination (normal seedlings). The seedling vigour index (Vi) was calculated as Vi = (mean root length + mean shoot length) × (percentage germination) (Abdul-Baki and Anderson, 1973). To determine the dry weight, seedlings from each treatment in the germination tests were wrapped in aluminium foil placed in an oven at 100°C. Twenty-four hours later, the dried seedlings were allowed to cool to room temperature and their weight was determined. In addition to tomato cv. Tanya, the effect of three selected essential oils on seed germination was also assessed on tomato cv. Cal-J and Roma VF, which are commonly grown in Tanzania. Evaluation of essential oils for production of healthy tomato transplants under screen house conditions. Healthy tomato seed samples of cvs Tanya, Cal-J and Moneymaker collected from tomato growers in the Iringa region (Tanzania), were inoculated and treated with essential oils as previously described. One hundred seeds per cultivar per essential oil and untreated control were taken randomly from the treated seeds and were sown in polyethylene plastic trays (56.5×26.5×6 cm) containing a mixture of forest soil, rice husks and farm yard manure (3:1:1). The trays were kept in a in a screenhouse under local climatic conditions (temperature ranging from 25 to 33°C and RH usually above 85%) throughout the growth period. Seven days after sowing, 40 seedlings per treatment of each cultivar (10 seedlings per replicate in four replications) were randomly selected and transplanted to polyethylene sleeves (6.5×9.0 cm) containing the same plant growth substrate mixture as previously described. The sleeves with the seedlings were placed on benches in the screenhouse under the same temperature and RH conditions. Twenty one days after sowing, the tomato seedlings were evaluated for BLS incidence and severity as previously described. In addition to disease incidence and severity, the height and weight of seedlings were also assessed. The height of the seedlings was determined by measuring the aerial part of the seedlings from the soil surface to the terminal node of the developing leaf. To determine the fresh weight of the aerial part of the seedlings, they were carefully cut at the base of the stem with a scissor and their weight was determined with a balance type Sartorius AG Gottingen, CP323S (Wagtech, UK). The experiment was repeated three times at different times from March to September 2010. Data analysis. In the in vitro assays, the average number of colony forming units of X. perforans on NA was calculated based on 3 replications. The colony count values in the re-evaluation of the essential oils that showed the best ability to inhibit BLS on NA during the initial screening tests were arranged in a completely ran-


domised design with three NA plates per treatment in three replications. The experiments conducted for the in planta evaluation of antibacterial activity were arranged in a completely randomised design with 16 plants in the preliminary in planta assays and 3×16 plants in the re-evaluation of the essential oils that showed the best ability to reduce BLS in tomato during the initial screening tests. The effect of selected plant essential oils on seed germination of tomato cv. Tanya, Cal-J and Roma VF was evaluated in experiments arranged in a completely randomized block design with 100 seeds per tomato variety in a block with four replications. The experiments on the evaluation of selected plant essential oil for the production of healthy tomato transplants (plant height, fresh weight, BLS severity and incidence) were arranged in a completely randomised block design with 10 plants per tomato variety in each block replicated four times. Data analysis for BLS severity, seed germination, seedling vigour, plant dry and fresh weight and plant height were done using Proc GLM. Mean separation tests were calculated with the Student-Newman-Kuels (SNK) using the Statistical Analysis System software (SAS version 9.1, SAS Institute, USA).

RESULTS

In vitro and in planta evaluation of antibacterial activity. The essential oils of rosemary (Rosmarinus officinalis), niaouli (Melaleuca viridiflora), eucalyptus (Eucalyptus globulus), clove (Syzygium aromaticum), thyme (Thymus vulgaris) and lemon (Citrus limon) totally inhibited the in vitro growth of X. perforans unlike distilled water (negative control) (Table 1). The inhibitory effect of these six essential oils was similar to that obtained when seeds were treated with copper sulphate (positive control). Essential oils from frankincense (Boswellia thurifera), cypress (Cupressus sempervirens), red cedar (Juniperus virgiana) and ylang-ylang (Cananga odorata) did not reduce the number of X. perforans colonies on NA (Table 1). Indeed, the number of CFU recovered from samples treated with these four essential oils on NA was similar to those obtained from seeds treated with SDW (negative control) (Table 1). The bacterial counts (4.2×103 CFU/ml) obtained from washings of seeds treated with bergamot oil (Citrus bergamia) was lower than those obtained from washings of seeds treated with SDW (Table 1). Although seed treatment with the essential oils of clove, thyme and lemon totally inhibited the growth of the BLS pathogen in the in vitro tests, they also inhibited the total number of plants emerged compared to the total number of seed sown (Table 1), even when the concentration was reduced to 1% (data not shown) that was the limit of oils in in vitro assays. Therefore, these essential oils were excluded


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Table 3. The effect of rosemary, eucalyptus and niaouli oils on the germination of seeds of three tomato cultivars commonly grown in Tanzania.

Treatment Controls Sterile distilled water Copper sulphate Untreated seed Oil source Rosemary Eucalyptus Niaouli Mean F test

DSe) (%)

Tomato cultivars Cal-J NS (%) ABS (%)

DS (%)

NS (%)

Roma VF ABS (%)

DS (%)

6.50a 5.00a 5.25a

2.50ab 2.50ab 2.50ab

91.50a 93.75a 92.00a

5.00a 2.75a 5.75a

3.50a 3.50a 2.25a

91.00a 89.75a 91.50a

6.00a 6.75a 6.00a

3.00a 3.50a 2.50a

4.50a 4.25a 5.25a 5.12 ns

3.25ab 2.75ab 5.25a 3.12 *

94.25a 92.75a 90.00a 92.36 ns

3.25a 5.00a 5.25a 4.5 ns

2.50a 2.25a 4.50a 3.08 ns

91.00a 91.00a 91.25a 91.91 ns

6.50a 7.00a 5.75a 6.30 ns

2.50a 2.67a 3.00a 2.87 ns

NSc) (%)

Tanya ABSd) (%)

91.00a 92.50a 92.25a 92.25a 93.00a 89.50a 91.75a ns

Essential oils were applied to tomato seeds at a concentration of 2% overnight at 25ºC shaken at 130 rpm. c) NS= normal seedlings. d) ABS = abnormal seedlings. e) DS = dead seeds. Mean followed by same letters in a column are not significantly different based on the SNK at P ≤ 0.05. Values represent the percent in 400 seeds. * =significant at 10%. ns = not significant.

from further studies. In agreement with the in vitro tests, the treatment of seeds with rosemary, niaouli, and eucalyptus oil significantly reduced the incidence and severity of BLS in tomato seedlings (Table 1). These results were similar to seeds treated with copper sulphate and pathogen-free seeds (untreated seed control). Differently from the in vitro experiments, the treatment of seeds with ylangylang, bergamot, cedar, cypress and frankincense significantly reduced the incidence and severity of BLS in seedlings compared with the negative control (tomato seeds inoculated with X. perforans and treated with SDW) (Table 1). The results obtained with the re-evaluation of the three best performing essential oils (rosemary, eucalyptus and niaouli) were in agreement with the preliminary assays (data not shown). In addition, the results showed that, the average height (12.75 cm), fresh weight (0.43 g) and width (1.54 mm) of the seedlings grown from seeds treated with SDW were significantly lower (P≤0.001) compared with those of seedlings from seeds treated with niaouli, eucalyptus and rosemary essential oils (Table 1). The effect of these essential oils on the growth of tomato seedlings was similar to that of copper sulphate treatment and untreated seeds (Table 1). Effect on seedling vigour and plant dry weight. The effect of essential oils of rosemary, niaouli and eucalyptus on seedling growth is summarised in Table 2. Treatment of disease-free seeds of cv. Tanya with essential oils of rosemary, niaouli and eucalyptus did not affect significantly (P≤0.05) the root and shoot length, vigour and dry weight of seedlings compared with seeds treated with SDW, copper sulphate and the untreated controls (Table 2).

Effect on seed germination. The effect of essential oils of rosemary, niaouli and eucalyptus at 2% concentration on seed germination of three commonly grown tomato cultivars in Tanzania is shown in Table 3. Results showed that there was no significant difference between the number of normal and abnormal seedlings when disease-free seeds were treated with these essential oils as compared to seeds treated with copper sulphate, seeds immersed in sterile distilled water and untreated seeds (Table 3). However, the number of dead seeds was significantly different (P≤0.1) for the cv. Tanya but not for Cal-J and Roma VF. Indeed, the highest number of dead seeds of cv. Tanya was recorded in the lot treated with essential oil of niaouli (5.25% of seeds were dead), although this value was not significantly different from those obtained with other treatments (Table 3). Further experiment on seed germination showed that the number of dead seed exposed to essential oil of niaouli was significantly (P≤0.05) higher in cv. Tanya than in Cal-J and Roma VF (data not shown) compared with that shown by rosemary and eucalyptus oils, copper sulphate, SDW and untreated seeds. Therefore, niaouli was not selected for further experiments. Effect on tomato transplants under screenhouse conditions. Seed treatment with the essential oil of rosemary and eucalyptus significantly (P≤0.001) reduced the BLS incidence and severity in the transplants compared with the negative control (seed treated with SDW) of all the three tested tomato cultivars and in all three experiments conducted (Table 4). The effect was similar to that obtained with seedlings from seed treated with copper sulphate and untreated seeds (disease-free) (Table 4). The effect of rosemary and eucalyptus oil on the growth (i.e. plant height and weight) of tomato trans-


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plants are shown in Table 5. In all three experiments, seed treatment with essential oils of rosemary and eucalyptus had no detrimental effect on seedling height compared with untreated seeds, regardless of the cultivar tested (Table 5). Indeed, the height of seedlings from seeds treated with the two essential oils was similar or significantly higher (P≤0.05) than that of seedlings from seeds treated with SDW, copper sulphate or untreated (Table 5). In most of the experiments, the height of seedlings from seeds treated with SDW was lower than that of seedlings obtained from the other treat-


ments (Table 5). With exception of cultivar Moneymaker seed treatment with rosemary and eucalyptus oils resulted in higher transplants compared with transplants obtained from seeds treated with SDW (Table 5). Similar results were obtained with the plant fresh weight (Table 5). Seed treatment with rosemary and eucalyptus oils had no negative effect on the seedling fresh weight compared with untreated seeds regardless of the cultivar tested (Table 5). Certainly, the weight of the seedlings obtained from the treatment of seeds with rosemary and eucalyptus oils was also similar or signifi-

Table 4. The effect of rosemary and eucalyptus oils in the control of BLS disease on transplants of three tomato cultivars under screen house conditions in Morogoro, Tanzania. Experiment 1 Disease Disease incidence severity (%) c) index d)

Experiment 2 Disease Disease severity incidence index (%)

Experiment 3 Disease Disease severity incidence index (%)

Variety

Treatment

Tanya

Controls Sterile distilled water Copper sulphate Untreated seed

45.00a

2.15a

82.50a

2.56a

65.00

2.18a

10.00b 0.00b

1.10b 1.00b

0.00b 0.00b

1.00b 1.00b

0.00b 0.00b

1.00b 1.00b

Oil source Rosemary Eucalyptus Mean F test

0.00b 0.00b 11.00 ***

1.00b 1.00b 1.25 ***

0.00b 0.00b 16.50 ***

1.00b 1.00b 1.31 ***

0.00b 0.00b 13.00 ***

1.00b 1.00b 1.24 ***

Cal-J

Moneymaker

c) Disease


70.00a

2.18a

80.00a

3.55a

62.50a

1.65a

0.00b 0.00b

1.00b 1.00b

0.00b 0.00b

1.00b 1.00b

0.00b 0.00b

1.00b 1.00b


0.00b 0.00b 14.00 ***

1.00b 1.00b 1.24 ***

0.00b 0.00b 15.00 ***

1.00b 1.00b 1.31 ***

0.00b 0.00b 12.50 ***

1.00b 1.00b 1.13 ***


65.00a

2.28a

67.50a

2.40a

72.50a

2.00a

0.00b 0.00b

1.00b 1.00b

0.00b 0.00b

1.00b 1.00b

0.00b 0.00b

1.00b 1.00b


0.00b 0.00b 13.00 ***

1.00b 1.00b 1.25 ***

0.00b 0.00b 13.5 ***

1.00b 1.00b 1.28 ***

0.00b 0.00b 14.50 ***

1.00b 1.00b 1.20 ***

incidence = number of seedling with bacterial leaf spot symptoms/total number of plants x 100%. d) Disease severity index based on Horsfall and Barrett (1945) scale of 1-6 with modification, where 1= no disease and 6 = > 50% BLS symptoms on tomato leaves. Mean followed by same letters in a column are not significantly different based on the SNK at P ≤ 0.05. ** *=significant at 1%.


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Table 5. The effects of two selected essential oils on the height and weight of transplants of three tomato cultivars under screen house conditions in Morogoro, Tanzania. Variety

Experiment 1 Height (cm) Weight (g)




19.66b 18.51b 18.41b

3.00b 3.00b 3.00b

17.58b 20.30a 21.15a

3.33b 3.95ab 3.93ab

14.72b 17.11a 17.36a

2.41b 2.72ab 2.36b

Oil sources Rosemary Eucalyptus Mean F test

23.07a 23.53a 20.60 ***

4.31a 4.4a 3.47 ***

19.60a 21.06a 19.93 ***

3.76ab 4.47a 3.89 **

17.75a 18.21a 17.03 ***

2.70ab 3.05a 2.65 **


23.02a 20.70ab 20.42b

4.14a 3.46a 3.30a

16.79c 18.25bc 18.33bc

2.32c 3.50b 3.35b

15.60c 17.34bc 18.43ab

2.69a 2.96a 2.91a


22.32ab 21.23ab 21.58 **

3.60a 3.88a 3.68 ns

20.22ab 20.50a 18.81 ***

4.23a 3.89ab 3.46 ***

19.40a 17.15bc 17.57 ***

3.42a 2.74a 2.94 ns


18.10b 22.56b 22.65b

2.85c 4.58ab 4.14bc

18.52a 19.00a 19.78a

3.36b 3.61ab 3.63ab

16.53b 18.10ab 19.52a

2.71b 3.22ab 3.51a


25.14a 21.63b 22.01 ***

5.65a 3.69bc 4.18 ***

19.10a 20.38a 19.35 ns

4.50a 3.84ab 3.79 **

18.53ab 19.55a 18.43 **

3.71a 3.71a 3.37 ***

Treatment

Tanya

Cal-J

Moneymaker

Mean followed by same letters in a column are not significantly different at P ≤ 0.05 based on SNK. ** = significant at 5%. *** = significant at 1%. ns = not significant.

cantly different (P ≤ 0.05) than the weight of seedlings from seeds treated with SDW copper sulphate, and untreated seeds (Table 5).

DISCUSSION

The preliminary evaluation of essential oils for seed treatment at 2% concentration to control X. perforans, one of the causal agents of BLS of tomato, showed that six out of eleven essential oils tested totally inhibited the growth of the BLS pathogen whereas five of them had little or no effect on the pathogen growth in vitro (Table 1). Rosemary, eucalyptus and niaouli oils significantly reduced the number of CFU of X. perforans on NA (Table 1). The antibacterial activity of these essential

oils had previously been reported in in vitro assays. For example, Trivedi and Hotchanadani (2004) and Moghtader and Afzali (2009) indicated that eucalyptus and rosemary oils have strong antibacterial activity against Klebsiella spp., Proteus spp., Pseudomonas spp., Escherichia coli, and Staphylococcus aureus. Similarly, niaouli showed a strong antibacterial effect against S. aureus (Christoph and Stahl-Biskup, 2001). The antibacterial activity was attributed to chemical compounds present in these oils such as eucalyptol (cineole) in eucalyptus oil (Trivedi and Hotchanadani, 2004); a-pinene, camphor, verbenone and 1,8-cineole in rosemary oil (Moghtader and Afzali, 2009); and a-pinene, limonene, 1,8-cineole, terpineol, nerolidol and viridiflorol in niaouli oil (Christoph and Stahl-Biskup, 2001). The antibacterial effect of the essential oils of rosemary and eu-


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calyptus oils were also reported to inhibit quorum sensing signals that play an important role in pathogenicity and antibiotic resistance in bacteria (Szabo et al., 2010). In the present study, the antibacterial effect of eucalyptus, niaouli and rosemary oils were seen both in in vitro and in planta assays. Indeed, these essential oils at 2% concentration reduced consistently the incidence and severity of BLS in tomato seedlings growing in both growth chamber and screenhouse (Table 1 and 4). The ability of eucalyptus, niaouli and rosemary oils to control BLS in planta, indicated that they might constitute potential alternatives to copper compounds against Xanthomonas spp. in tomato and similar hosts. In fact, there are many advantages of using essential oils in seed treatments for disease control, as many of these oils are produced from plants that are available locally in many areas of Tanzania and Africa in general, which make their use sustainable and cost effective. Moreover, essential oils are environmental friendly, little toxic to human and biodegradable and the low concentration required for seed treatment reduces the risk of environmental pollution. In terms of human toxicity, some of these essential oils, as is the case of eucalyptus oil, is indeed categorized as GRAS (Generally Regarded as Safe) by the Food and Drug Authority of USA and considered to be non-toxic (USEPA, 1993). Eucalyptus oil is also approved to be used as a flavouring agent in foods (≤5 mg/kg) by the European Union (Council of Europe, 1992). The use of essential oils in the control of bacterial pathogens is also suitable for organic crop production systems that currently lack efficient bactericides (Prakash and Roa, 1997). In addition to inhibit BLS in vitro and in vivo, treatment of tomato seeds with eucalyptus, niaouli and rosemary had no negative effect on seedling growth (Table 1 and 2). Some of these essential oils such as those from eucalyptus and rosemary have also been reported as green pesticides due to their non-toxicity to animals, bird and fish (Koul et al., 2008). The effect of these oils was similar to that of untreated tomato seeds, SDW and copper sulphate treatments. Furthermore, in the present study, the lowest height, weight and width of seedlings from inoculated seed treated with SDW (Table 3), indicated losses that may incur if no seed treatment is applied to infected seed. Although there was no significant difference between the number of dead seeds from the seed treatments used (Table 3), re-evaluation of essential oil from niaouli resulted in higher numbers of dead seeds in cv. Tanya compared with cv. Cal-J and Roma VF (data not shown) implying that the effect on seed germination of different essential oils may vary in different tomato varieties. In addition, seeds treated with eucalyptus and rosemary oils under screenhouse condition resulted in healthy transplants compared with seeds immersed in sterile distilled water controls (Table 4 and 5). Since seed treat-


ment with eucalyptus and rosemary oils did not have negative effects on the germination of seeds and growth of cvs Tanya, Cal-J, Roma VF and Moneymaker, they can be used for seed treatments of these popular tomato cultivars in Tanzania. In conclusion, the essential oils from eucalyptus and rosemary used as seed treatment at a concentration of 2% are very promising for the control of X. perforans in tomato. Their use of for seed treatment against xanthomonads in tomato will particularly be important against copper resistant strains reported in Tanzania (Shenge, 2006). Further studies will be required to investigate shelf life of the treatment to seeds, mode of action, analysis of the effective oil fractions in the control of bacterial leaf spot pathogens and the effects of these oils on tomato yield. Investigations on the interaction of the essential oils with hormones and phenolic acids which are responsible for the regulation of physiological processes during seed germination are also needed (Djanaguiraman et al., 2005). Further work also is required to investigate whether eucalyptus and rosemary oils can be used in the control of other seed-borne pathogens of tomato and in other crop systems.

ACKNOWLEDGEMENTS

The authors wish to thank the Danish Development Assistance (DANIDA) through ENRECA LIFE Project No. 731 for providing funds for this work.

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