Evaluation of acibenzolar-S-methyl and other low-toxicity products for control of rose powdery mildew (Podosphaera pannosa) in greenhouses

Abstract

In an attempt to find alternative products to classical fungicides, several products with low toxicity were tested against powdery mildew of roses. These products included resistance inducers (Bion, BABA, and ROS), potassium salts (Resistim, monopotassium phosphate), and seed extracts. The best results were obtained with acibenzolar-S-methyl (Bion). The utilization of Bion as prophylactic treatment, watered at a concentration 0.1–0.2 mg/ml, together with good cultural practices can be enough to effectively control powdery mildew on roses. Treatments with Resistim reduced the disease incidence, but not always significantly compared to the controls. None of the other products had effect on powdery mildew.

Keywords:

• ASM
• BABA
• Bion
• BTH
• grapefruit (C-pro)
• induced resistance
• Neemazal
• potassium phosphate
• Resistim
• ROS

Introduction

Rose powdery mildew, caused by Podosphaera pannosa, is one of the most destructive diseases of greenhouse grown roses (Rosa spp.). The use of resistant cultivars and good management of climatic conditions can keep powdery mildew at a low level. Still, the wide spread use of susceptible cultivars and difficulty in controlling climatic conditions in some periods of the year, make the use of fungicides necessary. In the case of ornamental crops, the total absence of visual damages is very important, and relative low levels of disease make the crop unmarketable. Thus, the use of considerable amounts of fungicides in the production of roses is inevitable. Some of the fungicides used against powdery mildew quickly loose efficiency because of resistance in the pathogen population (McGrath, 2001). This, and the wish to minimize the use of chemical products, leads to the search for alternatives to traditional fungicides.

Products that induce resistance in plants (elicitors) can reduce the severity of a pathogen attack, even if they do not prevent disease completely (Hammerschmidt et al., 2001). These products are of low toxicity and can help to obtain a good level of control in combination with other measures. Because these compounds act on the plants and not directly on the pathogen, the arising of resistant strains will take more time if it occurs at all. They could also decrease, if not completely avoid, the use of toxic fungicides. By reducing the use of fungicides, the use of inducers of plant resistance can also prolong the life span of the fungicides. Several of these products, like acibenzolar-S-methyl (BTH), β-aminobutyric acid (BABA), or reactive oxygen species (ROS), have been tested with good results against powdery mildew and other diseases (Hammerschmidt et al., 2001; Reignault & Walters, 2007; Beckers & Conrath, 2007). Several salt solutions have been used in the control of powdery mildew. Some of these compounds have both a preventive and a curative effect, and the preventive effect is due to the induction of resistance on plants (Reuveni et al., 1998). Application of phosphates can suppress lesions and eradicate conidia of powdery mildew in roses but also shows a preventive effect when applied before inoculations (Reuveni et al., 1994). Mineral and vegetal oils have been successfully used in the control of different powdery mildews, including rose powdery mildew (Pasini et al., 1997). Three modes of actions are described for oils: protective, curative, and antisporulative (Northover & Schneider, 1966). Plant extracts have been used against powdery mildew but the mode of action of these products is not well known. It is suggested that they can affect both the plant and the pathogen (Pasini et al., 1997; Toppe et al., 2007).

The aim of this study was to find alternative products for the control of powdery mildew of roses in greenhouse production. Special emphasis was made in checking the possibility of these products as prophylactic treatments by triggering induced resistance in plants and in finding products allowed to use in Norwegian greenhouse production.

Material and methods

Products tested

The products tested were: Bion 50 WG [Ciba, Basel, Switzerland; benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester (BTH)], Resistim (Brøste A/S, Lyngby, Denmark; Potassium phosphit [6.7 P 10.9 K] and plant betains), potassium phosphate (Sigma-Aldrich Co., St. Louis, MO, USA; KH₂PO₄), BABA (Sigma-Aldrich Co., St. Louis, MO, USA; BABA), C-Pro CE601(Citro Europa, grapefruit seed extract diluted and naturalized to pH 6.0–6.5. Raw material obtained from Citridall, Ripton, USA), NeemAzal-T/S (Trifolio-M, GmbH, Lahnau, Germany; neem oil), Dewasid Agri (DPI Chemical Ind., AS, Norway; Hydrogenperoxid, 3–12% + acetic acid, 10–30% + peracetic acid, 1–5%; 30–60%).

Cultural conditions

All the experiments were carried out with potted roses cv Pink Monte Rosa, considered as highly susceptible to powdery mildew by the growers. The plants (potted in 12 cm diameter pots) were received directly from a commercial nursery just after pruning. The trials were conducted in a free-standing greenhouse (2×1.5) m². The greenhouse had a heating and cooling system, and the temperature was set to 22°C day and 18°C night. Fluctuations in temperature between 17 and 27°C were recorded during the experiments. High-pressure sodium lamps gave supplementary light 18 h/day at 120 W/m² when natural light was insufficient. The set up for air humidity was changed during the experiments: 60% relative humidity (RH) before inoculation, 100% RH during inoculation (3 days), and 80% RH after inoculation. The recorded RH during the experiments fluctuated between 60 and 95%. The pots were watered manually every day with a complete nutrient solution with an electrical conductivity (EC) level of 2.5–3 mScm⁻¹. The nutrient solution was prepared using calcium nitrate, ammonium nitrate, and a fertilizer containing macro- and micro-nutrients.

Experimental design and treatments

Eight pots were used per treatment, each pot containing three plants. The experiments had a randomized block design, with one pot for each treatment per block. The newly pruned plants were placed in the greenhouse and allowed to grow for 5–7 days before they were treated. The first treatment was always done 5 days before inoculation, and in the cases where a second treatment was applied, it was done 13 days after the first. The different treatments can be seen in Tables I–III. The concentrations were chosen based on information available in the literature. When the products were applied by spraying, garden spray bottles were used.

Table I. Effect of different products against rose powdery mildew (Podosphaera pannosa) on cv Pink Monte Roso.

		Application date (31 January, before inoculation^a)		Disease severity^b (%)
Treatment	Product	Concentration/ml^c	Application way^d (10 ml)	13 February	20 February
1	Control (water)		Watering	23.3a	60.4a
2	Control (water)		Spray	25.8a	60.4a
3	Bion 50 WG	0.1 mg	Watering	6.7de	15.8d
4	Bion 50 WG	0.1 mg	Spray	0.8f	17.1d
5	Bion 50 WG	0.01 mg	Watering	22.9ab	45.4bc
6	Bion 50 WG	0.01 mg	Spray	17.1abcd	46.7bc
7	Resistim	0.1 ml	Watering	10.0bcde	42.9c
8	Resistim	0.1 ml	Spray	4.2def^e	42.1c
9	Resistim	0.05 ml	Watering	8.3de	55.4ab
10	Potassium phosphate	5 mg	Watering	22.1abc	45.0c
11	Potassium phosphate	5 mg	Spray	12.9bcde	52.1abc
12	Potassium phosphate	10 mg	Watering	20.8abcd	55.0ab
^aInoculation date: 5 February.
^bMean values with the same letters within a column are not significantly different (P<0.05).
^cConcentration of commercial products. Dilutions were made with distilled water.
^dTreatments were applied by drenching the pots or by foliar spray with 10 ml of solution per pot.
^eThe leaves were heavily damaged after the treatment. New growth of susceptible young leaves had merely started.

Table II. Effect of different products against rose powdery mildew (P. pannosa) on cv Pink Monte Rosa.

		First application (25 March, before inoculation^a)		Second application (7 April, after inoculation^a)		Disease severity^b (%)
Treatment	Product	Concentration/ml^c	Application way^d	Concentration/ml^c	Application way^d	7 April	14 April
1	Control (water)		Watering, 10 ml			15.0	36.2a
2	Control (water + Tween 20)	10^−4 ml (0.01%) Tween	Spray, 100 ml			13.8a	39.6a
3	Control (water )		Spray, 100 ml		Spray, 100 ml	6.3abc	31.2ab
4	Control (water + Tween 20)		Watering, 100 ml	10^−4 ml	Spray, 100 ml	7.9abc	38.3ab
5	Bion 50 WG	0.1 mg	Watering, 10 ml			1.7d	7.1d
6	Bion 50 WG	0.1 mg	Spray, 10 ml			3.7c	17.5c
7	Bion 50 WG	0.05 mg	Watering, 10 ml			7.5abc	22.5bc
8	Bion 50 WG	0.05 mg	Spray, 10 ml			4.1bc	25.8abc
9	Resistim	0.1 ml	Watering, 10 ml			6.3abc	23.7bc
10	Resistim	0.1 ml	Watering, 10 ml	0.1 ml	Watering, 10 ml	8.3ab	28.3abc
11	Resistim + Tween 20	0.01 ml + 10^−4 ml	Spray, 100 ml			5.8bc	21.7bc
12	Resistim + Tween 20	0.01 ml + 10^−4 ml	Watering, 10 ml	0.01 ml + 10^−4 ml	Spray, 100 ml	7.9abc	20.0
^aInoculation date: 30 March.
^bMean values with the same letters within a column are not significantly different (P<0.05).
^cConcentration of commercial products. Dilutions were made with distilled water.
^dTreatments were applied by drenching the pots or by foliar spray. The total amount of solution applied was 10 or 100 ml/pot.

Table III. Effect of different products against rose powdery mildew (P. pannosa) on cv Pink Monte Rosa.

		First application (18 February, before inoculation^a)		Second application (2 March, after inoculation^a)		Disease severity^b (%)
Treatment	Product	Concentration/ml^c	Application way^d	Concentration/ml^c	Application way^d	2 March	9 March
1	Control (water)		Watering, 10 ml			40.8a	74.2abc
2	Control (water)		Spray, 50 ml		Spray, 50 ml	45.8a	75.4ab
3	Bion 50 WG	0.2 mg	Watering, 10 ml			6.3c	30.0e
4	Bion 50 WG	0.1 mg	Watering, 10 ml			19.4b	52.5d
5	BABA	0.4 mg	Watering, 10 ml			37.1a	75.8ab
6	BABA	0.2 mg	Watering, 10 ml			35.8a	75.9a
7	C-Pro	2 mg	Watering, 10 ml			34.6a	74.2abc
8	NeemAzal	5 µl	Watering, 10 ml			37.9a	79.2a
9	NeemAzal	5 µl	Watering, 10 ml	1 µl	Spray, 50 ml	40.7a	75.0ab
10	NeemAzal	1 µl	Watering, 10 ml	1 µl	Spray, 50 ml	43.0a	68.8bc
11	Dewacid	50 µl	Spray, 50 ml			40.0a	77.1a
12	Dewacid	25 µl	Watering, 10 ml			45.4a	77.9a
13	Dewacid	25 µl	Watering, 10 ml	25 µl	Watering, 10 ml	42.5a	80.8a
14	Dewasid	5 µl	Spray, 50 ml	5 µl	Spray, 50 ml	37.1a	75.4ab
15	Bion 50 WG+Dewacid	0.05 mg+25 µl	Watering, 10 ml			23.8b	66.7c
^aInoculation date: 23 February.
^bMean values with the same letters within a column are not significantly different (P<0.05).
^cConcentration of commercial products. Dilutions were made with distilled water.
^dTreatments were applied by drenching the pots or by foliar spray. The total amount of solution applied was 10 or 50 ml/pot.

Inoculation and disease incidence

The inoculation was made with roses of the same cultivar, heavily infected with powdery mildew (“snowed plants”). The experimental plants were dusted and rubbed with the infected plants. The inoculation plants were then allowed to sporulate inside the greenhouse for 1 day and the procedure was repeated the day after. Assessment of powdery mildew incidence was made on two leaves per plant (six leaves per pot). Two assessments were made per treatment, the first 8 days and the second 15 days after inoculation. In the cases where two treatments were applied, the first assessment was done just before the second treatment. In the first assessment, the powdery mildew was usually only visible on one level of leaves, the third or fourth expanded leaf from the top. The infected leaf, or the one at the lowest level if there were several, was labeled. In the second assessment, this leaf was again taken into account together with the most heavily infected leaf. Infected leaf area (%) was recorded using a scale from 1 to 6: 1 = 0%, 2 = 0–1%, 3 = 2–5%, 4 = 6–24%, 5 = 25–50%, 6 = 51–100% (Prince, 1970; EPPO, 1987). Calculation of degree of infection was done following the equation of Townsend and Heuberger (1943):

P=infection degree (disease severity);

n=number of occasions at a certain level;

ν=infection levels 1–6;

Vmax = infection level 6; and

N=total number of leaves studied.

The data obtained were analysed and means were separated using ordinal logistic regression in Minitab.

Results

The results from the first experiment are shown in Table I. In the first assessment, all treatments with Resistim (7, 8, and 9) and the treatments with the highest concentration of Bion (3 and 4; 0.1 mg/ml) showed significant decrease in disease severity compared to the inoculated controls. For many of the plants treated with Bion, no infected leaves were detected at all. The reduction of disease for treatments 3 and 4 was 97 and 71%, respectively. The young leaves of plants sprayed with Resistim at a concentration of 0.1 ml/ml (treatment 8) were scalded. The low percentage of disease in this treatment was due to the absence of sensitive leaves at the moment of inoculation. The two treatments with Resistim where the leaves were not damaged (7 and 9), showed a disease reduction of 57 and 64%, respectively. In the second assessment, the treatments with Bion and the treatments with the highest concentration of Resistim (7 and 8; 0.1 ml/ml) showed a significant reduction in disease severity compared to the inoculated control. The higher concentrations of Bion had a disease reduction of approximately 70% and the higher concentrations of Resistim of approximately 30%. The treatments with Resistim at the highest concentrations showed no significant difference compared to the lowest concentrations of Bion, and the disease reduction for all these treatments were equal to or lower than 30%. The method of application, watering or spraying, did not influence the results. The results with potassium phosphate were in general poorer and more erratic than with Bion or Resistim.

The results of the second trial are shown in Table II. The disease severity in the first assessment was low and in consequence it was more difficult to see differences between the treatments. The only treatment that showed significant decrease in disease severity compared to the inoculated control was treatment 5 (watering with 0.1 mg/ml Bion). In the second assessment, all treatments showed a tendency to be better than the controls, however, the only treatments with significant difference was treatments 5 and 6 (Bion 0.1 mg/ml, watering and spraying). Applying Resistim twice (treatments 10 and 12) or the addition of Tween (treatments 11 and 12) did not improve the performance of the product.

In the third experiment, Bion, BABA, C-Pro, NeemAzal, Dewasid and a mixture of Bion and Dewasid were tested (Table III). In the first assessment, the two treatments with Bion at concentrations 0.2 and 0.1 mg/ml (treatments 3 and 4) showed a significant differences in disease severity compared to the controls. The disease reduction was 85 and 53%, respectively. The mixture of Bion and Dewasid (treatment 15) also significantly lowered the level of disease compared to the controls and reduced the severity of disease by 42%. None of the treatments with Dewasid alone showed a significant reduction in disease severity. In the second assessment, only the two treatments with Bion showed significant differences compared to the two inoculated controls with a reduction in disease of 60 and 29%.

Discussion

The results in our experiments, applying BABA or potassium phosphate as drenching 5 days before inoculation, were bad or inconclusive. BABA was reported to reduce to some degree powdery mildew of cucumber (Vogt & Buchenauer, 1997) and melon (Bokshi et al., 2005), and potassium phosphate has been used as a protective treatment for powdery mildew on roses by spraying plants 4 days before inoculation (Reuveni et al., 1994). We could not observe any effect of C-Pro when applied as drenching before inoculation. C-Pro showed effect against powdery mildew of cucumber and roses when sprayed directly on the fungus and it was suggested that part of the effect could be due to induced resistance (Toppe et al., 2007). We did not observe any effect of neem oil on powdery mildew either when applying it before inoculation or directly on the fungus. However, neem oil is reported to reduce powdery mildew on roses (Pasini et al., 1997).

Dewasid is a product available in Norway that contains ROS. We tested this product alone or in combination with Bion (salicylic acid [SA] pathway activator). It did not have an effect on powdery mildew on roses when using it as a spray or by drenching alone. When mixed with Bion it significantly reduced the disease severity, but the effect seems to be only due to the presence of Bion. Oxycom is a product containing ROS and SA and it is reported to provide disease control in different crops and against several pathogens (Reignault & Walters, 2007). The utilization of ROS alone can have some effect but there is a synergism between ROS and SA (Blee et al., 2004).

In our experiments, Resistim showed some effect against powdery mildew. The effects of Resistim when applying the product on roots or leaves before inoculation suggest some degree of induced resistance. A second application did not improve the effect, but maybe a higher concentration could have improved the results. Application with a dispersant directly on the fungus did not improve the results compared to applying it to the roots. Resistim was first applied at a concentration 0.1 ml/ml (10 ml/pot) both by drenching and spraying. This concentration damaged the leaves of the plants when applied as a spray. Later, Resistim was applied at a concentration 0.01 ml/ml (100 ml/pot) and no damages were observed. Resistim contains potassium phosphit and plant betains, but the exact composition of the product it is not known. It is commercialized as a fertilizer that stimulates natural plant defenses and is easily available and commonly used in many greenhouses in Norway. Low dose applications of betains in plants are reported to enhance plants to resist stress conditions (Blunden, 2003), and phosphits (phophonates) are used as fungicides against oomycetes. The effect seems to be both direct on pathogens and indirect by stimulating plant defenses, and it has also been proved that it has some effect on other pathogens than oomycetes (Guest & Grant, 1991).

In our experiments, significant reduction of powdery mildew on roses was observed for all the treatments with Bion. A positive correlation was observed between concentration of Bion and effect on the disease. The effect of Bion was still evident 2 weeks after inoculation, meaning 20 days after application. No phytotoxicity was observed at the concentrations used in these experiments. However, when accidentally a very high concentration (100 mg/ml) was applied in a pilot experiment, the plants had stunted growth, produced whitened shoots and the leaves yellowed. Whitening of chrysanthemum apical leaves and inhibited plant growth has been reported by using a concentration 4.8 mM BTH (D'Amelio et al., 2010). Reduction of powdery mildew by use of BTH has been reported for different crops including monocotyledons (Görlach et al., 1996; Wiese et al., 2003) and dicotyledons (Bokshi et al., 2003, 2008; Achuo et al., 2004; Borkowski & Szwonek, 2004; Hukkanen et al., 2007). The utilization of 2,6-dichloroisonicotinic acid, an elicitor with a similar mode of action as BTH, reduced the development of rose powdery mildew (Hijwegen et al., 1996). Different responses between cultivars were observed, but in all cases disease reduction was achieved.

All the products tested in these experiments have earlier been reported to have effect on powdery mildew on different host plants. However, in our study, many of them showed no effect on powdery mildew of roses with the methods of application and concentrations used.

The utilization of Bion as prophylactic treatment, watered at a concentration 0.1–0.2 mg/ml, together with good cultural practices can be enough to effectively control powdery mildew on roses. In the cases of severe attacks, the utilization of conventional fungicides could be necessary. The effect of Resistim was not as good as the effect of Bion in this study, but the use of Resistim watered at a concentration 0.1 ml/ml or sprayed at a concentration 0.01/ml, could help to mitigate the incidence of the disease.

Acknowledgements

This work was funded by the Norwegian Research Council. We thank Widerøe nursery for providing plant material for all the trials and the collaboration from technicians at Centre for Plant Research (SKP), Norwegian University of Life Science (UMB) and Norwegian Institute for Agricultural and Environmental Research (Bioforsk).