Biological control of fruit rots on strawberry and grape by BOTRY-Zen

Abstract

Fruit rots are some of the most important diseases of grape and strawberry. With recent public concern regarding pesticide residues on fruit, there is a need for alternative disease management practices that will reduce the risk to consumers. The main aim of this study was to investigate the effectiveness of the commercial product BOTRY-Zen (active ingredient Ulocladium oudemansii [U3 strain]) to control strawberry and grape fruit rots. BOTRY-Zen at 6 g/L reduced significantly the percentage of rotted fruit in comparison with the untreated control. However, its effectiveness was significantly less than that of the fungicide Switch 25/37.5 WG (fludioxonil: cyprodinil) at a rate of 1 g/L (recommended by producer). There was no significant difference between the BOTRY-Zen at rate of 4 g/L and untreated plants. The results of this study showed that the commercial product BOTRY-Zen, at 6 g/L, could be a useful tool to control fruit rots of strawberry and grape in biological fruit production systems.

Keywords:

• Botrytis cinerea
• fungal pathogens
• Fragaria
• Ulocladium oudemansii
• Vitis

Introduction

Many fungi are capable of rotting mature or near mature fruits of strawberry and grape. Fungi of the genus Botrytis are mainly responsible for fruit rots in strawberry and grape. Other fruit rots of minor importance on grape and strawberry are caused by fungi of the genera Colletotrichum, Rhizopus, Aspergillus and Alternaria (Latinovic et al. 2012). Under favourable environmental conditions for disease development, serious losses can occur. The diseases are most severe during years with prolonged rainy and cloudy periods during bloom or harvest.

Disease control is achieved primarily through the use of fungicides, although orchard sanitation is an integral part of the management programme for this disease. Pre- and post-harvest losses of strawberry and grape fruits may be significant in some years, and fungicide treatment of fruit during the growing season as well as after harvest is routine. However, with recent public concern regarding pesticide residues on fruit, there is a need for alternative disease management practices that will reduce the risk to consumers.

Many fungi, yeasts and bacteria have been found to be capable of controlling fruit rot diseases, often due to the production of enzymes and antibiotics. Donmez et al. (2011) showed that antagonistic bacterial strains Bacillus C6, Brevibacterium MFD-47, Enterobacter MFD-81 and Pantoea MFD-232 inhibited Botrytis cinerea and they have a potential use in sustainable strawberry production. Calvo-Garrido et al. (2012) reported that Candida sake consistently provided effective control of botrytis bunch rot in grape under different meteorological and disease pressure conditions, thereby improving its potential for future commercial applications.

The active ingredient of the commercial product BOTRY-Zen, Ulocladium oudemansii (U3 strain), is a naturally occurring soil fungus existing as a saprophyte of dead and decaying plant matter. As a biofungicide, it is intended to protect fruit and vegetable crops and ornamental plants from plant pathogenic diseases by competing for the same ecological niches (senescent plant material) and nutrients. The Environmental Protection Agency (EPA) of the United States (PC Code: 102111) has determined that Ulocladium oudemansii (U3 strain) presents no issues of toxicological, ecological or environmental concern.

The aim of this study was to investigate the effectiveness of the biopesticide BOTRY-Zen (BOTRY-Zen Ltd, Dunedin, New Zealand) to control fruit rots in strawberry and grape.

Materials and methods

The application equipment used was a backpack sprayer (Boom ID–GR T19cr) with a spray volume of 1000 L/ha. The date of applications and the application conditions are presented in Tables 1 and 2. Twenty unsprayed bunches of grapes were transferred to the laboratory of plant pathology, Alexander Technological Educational Institute of Thessaloniki, to investigate the pathogens causing fruit rot on grape in 2012 and again in 2013. A total of 300 rotted grapes were collected from the bunches in each year. To isolate the pathogen (one isolation from each diseased fruit), small pieces, about 2 mm in diameter, were taken from the margin of rotted tissues and placed on acidified potato dextrose agar (2.5 mL 85% lactic acid/L nutrient medium) in Petri dishes of 55 mm in diameter, under aseptical conditions. The plates were incubated at 23 °C for 5 to 7 days, and the isolated pathogens were stored in a growth chamber until use. Identification of fungi was carried out by studying the morphology of the colony (mycelium) and reproductive organs (conidiophores, conidia, etc.) using taxonomic keys.

Table 1 Application date and application conditions in experimental field located in Svoronos, Pieria (strawberry).

2012
Date of application	9/4/2012	23/4/2012	13/5/2012
Start application (hh:mm)	10:00	11:00	10:00
Air temperature (°C)	25.7	28.2	27.3
Relative humidity (%)	42.2	42.1	43.4
Clouds cover (%)	80	30	0
Crop GS stage BBCH	Min = 14, Max = 55	Min = 65, Max = 73	Min = 81, Max = 82
Plant height	0.1–0.2	0.3–0.35	0.35–0.45
Wind speed (m/s)	0	0	0
2013
Date of application	13/4/2013	28/4/2013	13/5/2013
Start application (hh:mm)	9:00	10:00	11:00
Air temperature (°C)	13.2	15.6	19.9
Relative humidity (%)	38.5	46.9	39.4
Clouds cover (%)	0	50	0
Crop GS stage BBCH	Min = 16, Max = 40	Min = 55, Max = 65	Min = 78, Max = 82
Plant height	0.2–0.25	0.3–0.4	0.45–0.5
Wind speed (m/s)	0	0	0

Table 2 Application date and application conditions in experimental field located in Stantza Naoussa, Imathia (grape).

2012
Date of application	22/8/2012	5/9/2012	11/9/2012	18/9/2012	25/9/2012
Start application (hh:mm)	7:45	8:00	8:30	7:45	8:30
Air temperature (°C)	18.5	19.7	17.1	19.3	20.3
Relative humidity (%)	76.5	82.5	80	80	80.2
Clouds cover (%)	0	0	0	80	60
Crop GS stage BBCH	Min = 78, Max = 79	Min = 81, Max = 81	Min = 83, Max = 83	Min = 83, Max = 83	Min = 83, Max = 83
2013
Date of application	18/8/2013	25/8/2013	3/9/2013	10/9/2013	17/9/2013
Start application (hh:mm)	7:30	7:30	7:30	7:30	7:30
Air temperature (°C)	19.4	20.1	18.6	17.3	16.5
Relative humidity (%)	43.6	45.7	53	51	73
Clouds cover (%)	30	0	0	0	65
Crop GS stage BBCH	Min = 75, Max = 78	Min = 80, Max = 81	Min = 83, Max = 83	Min = 83, Max = 83	Min = 83, Max = 83

In all the experiments, two concentrations of BOTRY-Zen (5.7 × 10⁹ cfu/g) were evaluated: 4 g BOTRY-Zen/L (recommended by producer) and 6 g BOTRY-Zen/L. The higher concentration of BOTRY-Zen (6 g/L) was used because preliminary laboratory work had shown that the dose recommended by the producer was not very effective (T. Thomidis, unpubl. data).

The experiment for strawberry (cv. June Bearing) was conducted in a commercial field in Svoronos. The dates and growth stages (BBCH) of plants sprayed with BOTRY-Zen are presented in Table 1.

The experiment for grape (cv. Xinimavro Naoussa, 8-year-old) was conducted in a commercial field in Strantza Naousas, in the Prefecture of Imathia. The first application of BOTRY-Zen on grape was conducted at the growth stage of development of fruits (BBCH scale: 75–79) (Tables 1 and 2) during which the grapes are susceptible to B. cinerea under the climate conditions of northern Greece. This is slightly later than the 2%–5% capfall (c. GS60–61) recommended in best practice guidelines from the producer. Plants were grown under commercial cultural practices and no fungicide was applied against fruit rots.

The experimental design was a completely randomized block design. There were five replicates, each with 10 plants. In addition, there were 10 plants sprayed with the mix fludioxonil: cyprodinil at a rate (1 g/L) recommended by the producer (Switch 25/37.5 WG, Syngenta Hellas) and 10 unsprayed plants used as control. Results for strawberry were collected on 15 and 24 May in the first year of experiments (2012) and 18 and 25 May in the second year (2013), and for grape on 28 September in the first year of experiments (2012) and 20 September in the second year (2013). Fifty fruits per bunch were collected randomly from each treatment at harvest time and the percentage of rotted fruits was recorded.

Data for fruit rots expressed in percentages were analysed after angular transformation to active normality (data presented in the tables are back-transformed). For testing of differences, the Wald chi-square test was applied at the significant level a = 0.05.

Results and discussion

Species of the genus Ulocladium contain both plant pathogens and food spoilage agents. Previous works have shown that the biocontrol agent directly attacks the plant pathogen by secreting lytic enzymes such as chitinase, -1,3-glucanase, cellulase and proteases (Haran et al. 1996), and these enzymes hydrolyse the pathogen’s cell wall components such as chitin, glucan, cellulose and proteins successfully limiting the growth of fungal pathogens (Carsolio et al. 1999). Aggressive colonization of necrotic bunch trash tissues by U. oudemansii early in the season is the primary mode of action (Shorten et al. 2003). The results of this study showed that BOTRY-Zen at a rate of 6 g/L had reduced significantly the percentage of fruit rots in grape and strawberry on both dates of assessment (Table 3). In contrast, there was no significant difference between BOTRY-Zen at a rate of 4 g/L and unsprayed plants. Elmer et al. (2005) found that the product BOTRY-Zen was effective to control B. cinerea in grape. Reglinski et al. (2005) suggested the combination of chemical elicitor, 5-chlorosalicylic acid (5CSA) and U. oudemansii to control Botrytis bunch rot in Chardonnay grapevines.

Table 3 Comparison of the percentage of strawberry or grape rotted after treatment with BOTRY-Zen, Switch 25/37.5 WG and non-treated fruit.

		Strawberry				Grape
		Date of assessment				Date of assessment
		2012
Treatments	Rate (g/L)	15/5		24/5		28/9
Unsprayed control	–	2.81	a	5.42	a	5.03	a
BOTRY-Zen	4	2.09	a	5.75	a	4.53	a
BOTRY-Zen	6	0.94	b	2.78	b	3.36	b
Switch 25/37.5 WG	1	0.22	c^z	0.92	c	0.55	c
		2013
		19/5		25/5		20/9
Unsprayed control	–	4.77	a	14.10	a	9.44	a
BOTRY-Zen	4	5.22	a	11.45	a	7.79	a
BOTRY-Zen	6	3.70	b	7.89	b	4.65	b
Switch 25/37.5 WG	1	0.50	c^z	0.77	c	1.80	c

^zValues in the same column followed by the same letter are not significantly different according to Wald test (P < 0.05).

Isolation of pathogens from the control grape and strawberry fruits showed that the fungus B. cinerea was the dominant pathogen in 82% (246 of 300 isolates) and 87% (261 of 300 isolates) of cases, respectively. Fungi of the genera Colletotrichum, Rhizopus, Alternaria and Aspergillus were also isolated from rotted fruits. Previous works also showed that the fungus Ulocladium atrum can effectively control B. cinerea in many plant species (Kohl et al. 1995; Gerlagh et al. 2001). Kohl (2004) reported that U. atrum combines a high competitive colonization ability with a superior ecological competence. According to Köhl et al. (2004), U. atrum could be used to control Alternaria radicina in the seed production of carrots. Yun et al. (2007) isolated an antifungal cyclopeptolide from antagonist U. atrum which exhibited potent antifungal activity against B. cinerea and moderate activity against Alternaria alternata and Magnaporthe grisea. However, Kessel et al. (2002) reported that competition for resources is sufficient to explain the dynamic interactions between B. cinerea and U. atrum. This information can be used as a tool to optimize the dose and timing of U. atrum applications providing effective biological control of B. cinerea.

This study showed that the commercial product BOTRY-Zen could be a useful tool to control fruit rots of strawberry and grape in a biological fruit production system. However, a spray programme including BOTRY-Zen and fungicide application is recommended in a biological fruit production system to reduce the percentage of fruit rots. This product could also be used in an integrated fruit production system to control fruit rot diseases during the harvesting or for post-harvest applications when no other pesticide is allowed. Although a dose of 4 g BOTRY-Zen/L is recommended by the producer, the results of this study showed that a higher dose (6 g BOTRY-Zen/L) is needed for commercial uses. Further work is needed to determine the effectiveness of higher doses of BOTRY-Zen than those used in this study.

Acknowledgements

This work was funded by O.P. Competitiveness and Entrepreneurship (EPAN ΙΙ), ROP Macedonia—Thrace, ROP Crete and Aegean Islands, ROP Thessaly—Mainland Greece—Epirus, ROP Attica.