Managing Botrytis blossom blight of wild blueberry through field sanitation, lime sulfur and Trichoderma application

Abstract

Botrytis blossom blight is an important disease of wild blueberries (Vaccinium angustifolium) and causes substantial yield loss annually. Field trials that involve primary inoculum reduction and disease reduction techniques were conducted for the 2015/2016 and 2019/2020 growing seasons. The aim of this study was to evaluate the single and combined effects of thermal treatment, calcium polysulphide and Trichoderma harzianum T-22 application on Botrytis blossom blight development, physical plant structure and harvestable yield over the 2-year production cycle of the crop. The treatments were such that fields were thermally treated in May after pruning, followed by calcium polysulphide application in November 2015 and 2019. Trichoderma harzianum was applied in June 2016 and 2020 as single and combined treatments. Thermal treatment + calcium polysulphide + T. harzianum and thermal treatment + calcium polysulphide combinations reduced Botrytis blossom blight incidence and severity by over 50% and 74%, respectively, compared with the untreated control in both seasons. Plots receiving only the thermal treatment and calcium polysulphide as well as the calcium polysulphide + T. harzianum combination showed reductions in disease incidence of over 51, 38 and 81%, and reductions in disease severity of 55, 39 and 68%, respectively, compared with the untreated control. Plots that received thermal treatment combined with calcium polysulphide had taller stems, and a higher number of floral and vegetative buds. Results from this study have demonstrated that Botrytis blossom blight in wild blueberry fields can be adequately reduced when calcium polysulphide is used tandem with thermal treatment and biofungicides.

Résumé

La pourriture grise des fleurs est une importante maladie des bleuets sauvages (Vaccinium angustifolium et V. myrtilloides) et cause des pertes substantielles chaque année. Des essais au champ impliquant la réduction de l’inoculum primaire et des techniques de réduction de la maladie ont été menés pendant les saisons de croissance de 2015-2016 et de 2019-2020. Le but de cette étude était d’évaluer les effets individuels et combinés du traitement thermique, du polysulphure de calcium et de l’application de Trichoderma harzianum T-22 sur le développement de la pourriture grise des fleurs, la structure physique de la plante et la production récoltable durant le cycle productif de deux ans de la culture. Les traitements ont été tels que les champs ont d’abord été traités thermiquement en mai, après la taille, puis, en novembre 2015 et 2019, on leur a appliqué le polysulphure de calcium. Trichoderma harzianum a été appliqué en juin 2016 et 2020 comme traitement unique, puis combiné. Des combinaisons de traitement thermique + polysulphure de calcium + T. harzianum et de traitement thermique + polysulphure de calcium ont réduit l’incidence et la gravité de la pourriture grise par plus de 50% et de 74%, respectivement, comparativement au témoin non traité, et ce, durant les deux saisons. Les parcelles traitées uniquement avec la chaleur et le polysulphure de calcium de même qu’avec la combinaison de polysulfure de calcium + T. harzianum ont affiché des réductions de l’incidence de la maladie de plus de 51, 38 et 81% ainsi que de la gravité de la maladie de 55, 39 et 68%, respectivement, comparativement au témoin non traité. Les parcelles qui ont reçu le traitement thermique combiné au polysulphure de calcium comportaient de plus longues tiges et un plus grand nombre de bourgeons floraux et végétatifs. Les résultats de cette étude ont démontré que la pourriture grise des fleurs dans les champs de bleuets sauvages peut être adéquatement maîtrisée lorsqu’on utilize le polysulphure de calcium combiné au traitement thermique et aux biofongicides.

Keywords:

• biofungicide
• Botrytis cinerea
• lime sulphur
• pruning
• Trianum P
• Trichoderma harzianum
• Vaccinium

Mots clés:

• Botrytis cinerea
• biofongicide
• taille
• sulfure de calcium
• Trianum P
• Trichoderma harzianum
• Vaccinium

Introduction

The wild blueberry plant (Vaccinium angustifolium and V. myrtilloides) is a perennial low-growing shrub native to North America. Wild blueberry fields are developed from naturally occurring native plants when the vegetation cover of forested areas or abandoned farmlands are removed (Yarborough 2009). Commercial fields are managed on a 2-year cycle (vegetative year and the bearing or crop year) with the shoot being pruned every other year to maximize berry yield, and ease of mechanical harvest (Eaton et al. 2004).

Botrytis blossom blight caused by the fungus Botrytis cinerea Pers.:Fr is one of the most important diseases of wild blueberry and can be a serious problem during prolonged wet periods (Choquer et al. 2007; Walker et al. 2015). Historically, Botrytis blossom blight has been a problem in coastal areas where extended periods of rainfall and fog provide a suitable environment for B. cinerea infection (Lambert 1990). In addition, the frequent wetness periods associated with the maritime climate in places such as Nova Scotia, Canada, create suitable environmental conditions for B. cinerea infections to occur. Infected wild blueberry flowers show a brown, water-soaked appearance which spreads to cover the whole flower. Dead flowers are mostly covered with the characteristic dense greyish mycelia and of B. cinerea spores. Infections may move through the flowers quickly and often destroy the entire inflorescence (Hildebrand et al. 2001).

Botrytis cinerea has unique characteristics that enable it to survive for many years once established in a field. Although B. cinerea is a necrotrophic pathogen, it is capable of growing and reproducing on dead, senescent plant tissues and debris as a saprophyte. The fungus overwinters as sclerotia and dormant mycelia on weeds and plant debris within and outside the field (Elmer and Michailides 2007). During wet weather conditions with high humidity (> 90%) and moderately cool temperatures (15–20°C), especially in the spring and early summer, the overwintering structures germinate and produce conidia which infect blueberry plants (Delbridge and Hildebrand 1997; Smith 1998). Floral tissues are most susceptible, but infections can occur on other plant parts such as leaves (Delbridge and Hildebrand 1997).

Over the years, the management of Botrytis blossom blight has been achieved primarily through the application of conventional fungicides such as Switch® (cyprodinil and fludioxonil), Luna Tranquility® (fluopyram and fyrimethanil) and Pristine® (boscalid and pyraclostrobin) applied on a 7- to 10-day schedule with two to three fungicide applications per cropping season during bloom (Percival 2013). However, this control method is gradually becoming less effective due to the development of resistance among the pathogen population (Abbey et al. 2019). In addition, increased flower densities (38 million flowers·acre⁻¹ in 1994 to more than 150 million flowers·acre⁻¹ due to improved production practices such as nutrient management) which provide abundant susceptible tissues for pathogen proliferation (Percival 2013), and a strict maximum residue limit (MRL) on the international market raise concerns about the ability to effectively manage Botrytis blossom blight while meeting MRL conditions. Given this, there is a need to develop alternative approaches that will help address the current disease management challenges. Adoption of integrated disease management practices involving field sanitation, application of caustic product and biofungicide could prove effective for the management of Botrytis blossom blight and produce berries with limited/no chemical residue.

Field sanitation including the removal and/or destruction of infected plant tissue and debris is encouraged for the management of diseases caused by B. cinerea (Bika et al. 2021; Würz et al. 2020). However, the unique nature of wild blueberries makes it impossible to achieve field sanitation during the cropping season. Pruning/field sanitation can only be achieved at the beginning of the 2-year production cycle on commercial fields through mowing down or thermally treating vegetative shoots. Between these two pruning methods, the application of thermal treatments using oil-fired burners and/or straw have been found to reduce overwintering B. cinerea inoculum, and often reduce Botrytis and Monilinia blight (Monilinia vaccinii-corymbosi) (Lambert 1990; McGovern et al. 2012) and other pests and diseases (Jensen and Yarborough 2004; Yarborough 2004) in wild blueberry fields. Additionally, thermal treatment resulted in taller plants with greater yield (Ismail et al. 1981).

Calcium polysulphide (lime sulphur) has been found to reduce diseases such as scab (Venturia pirina) in pear by 96% (Jamar et al. 2017). In apple, a combination of calcium polysulphide and Regalia biofungicide, a plant extract (Reynoutria sachalinensis), reduced apple scab (Venturia inaequalis) and cedar apple rust (Gymnosporangium juniperi-virginianae) in four of five test years (DeLong et al. 2018). In highbush blueberries, calcium polysulphide (applied before bud initiation and some formative buds, dormant) has been reported as a disinfectant for Botrytis blight management (Bettiga 2013; Longstroth and Miles 2018). Furthermore, a dormant calcium polysulphide application was reported to reduce B. cinerea sclerotia by over 70% in grapes (Bettiga 2013).

The management of B. cinerea with biological control agents (BCAs) and other natural compounds instead of conventional fungicides has received enormous attention for over two decades because these are less damaging to the environment, and their complex mode of action decreases the risk of resistance development (Nicot et al. 2016). Among the known BCAs, Trichoderma spp. is one of the most studied and extensively used (Elad 2000; Barakat and Al-Masri 2017; Halifu et al. 2019). Over the years, significant control of B. cinerea diseases by T. harzianum has been reported on different crops in many studies (You et al. 2016; Barakat and Al-Masri 2017; Li et al. 2020). For example, T. harzianum T-22 was very effective against B. cinerea when applied either before or after infection of strawberry leaves and provided over 40% disease reduction (Robinson-Boyer et al. 2009). Barakat and Al-Masri (2017) reported up to a 45% reduction of Botrytis blight on detached strawberry leaves treated with T. harzianum Jn14. In addition, studies have shown that Trichoderma spp. enhance nutrient uptake, stabilize soil nutrients, and promote plant development (Stewart and Hill 2014). Halifu et al. (2019) reported a significant increase in seedling biomass when T. harzianum E15 and T. virens ZT05 were applied on Mongolian pine (Pinus sylvestris var. mongolica).

The disease control capabilities of field sanitation (thermal treatment), calcium polysulphide and T. harzianum have been studied on several crops. However, none of these products and treatments has been tested in wild blueberry, despite its importance and unique production system. This study was therefore conducted to investigate the main and combined effects of thermal treatment application, calcium polysulphide and T. harzianum applications on Botrytis blight and plant development in a commercial wild blueberry field.

Materials and methods

Site selection and experimental design

Field trials were conducted during the 2015–2016 and 2019–2020 growing seasons in a commercial wild blueberry field over the 2-year production cycle of the crop. The trials were set up at Debert, Nova Scotia (Ns) (coordinates = 45°26ʹ35.65 N, 63°27ʹ5.69 W) in May of 2015 and 2019 using a randomized complete block design (RCBD) with five replications. A plot size of 4 × 4 m with 2 m buffers between plots was used. The field was equipped with a Watchdog® model 2700 weather station (Aurora, IL) to monitor air temperature, relative humidity, leaf wetness, wind speed and direction at 15 min intervals.

Treatment combination and application

The treatments were (±) thermal treatment (±) calcium polysulphide (lime sulphur) (Loveland Products, ON) and (±) Trichoderma harzianum T-22 (Trianum P®) (Koppert Biological Systems, ON). The treatment combinations were 1) Untreated control, 2) Thermal treatment plus Calcium polysulphide plus T. harzianum, 3). Thermal treatment plus Calcium polysulphide, 4). Thermal treatment plus T. harzianum 5). Thermal treatment, 6) Calcium polysulphide plus T. harzianum 7) Calcium polysulphide and 8) T. harzianum Thermal treatment was done on the 22nd and 2nd of May 2015 and 2019, respectively after pruning using a Weed Dragon Model® VT2-23SVC 100000 BTU propane burner. The calcium polysulphide was applied at a rate of 11.25 L ha⁻¹ on the 25th and 21st of November 2015 and 2019 respectively when two thirds of the leaves had dropped and T. harzianum was applied at 3.0 g m⁻² (10⁹ CFU per gram of dry weight) on 26th May 2016 and 4th June 2020 respectively when plants were between bud break and pre-bloom growth stage. The calcium polysulphide and T. harzianum were applied using a Bell spray Inc.® hand-held carbon dioxide pressurized research sprayer with a 2 m boom equipped with four Tee Jet Visiflow 8003VS nozzles at a pressure of 220 kPa and application carrier volume of 250 L ha ⁻¹.

Disease assessment

Before bloom in the cropping year (May 2016 and 2020) ten blueberry stems were randomly selected from each plot for the assessment of incidence of Botrytis stem canker. Fifteen stems were also selected at mid-bloom (between 40–50% bloom) and full bloom for assessment of disease development on floral tissues and physical development parameters. The stems were cut as close to the base as possible diagonally at 20 cm intervals along a 4 m line transect in each plot. Stem samples were placed in plastic bags and brought to the laboratory for examination of Botrytis blight development (incidence and severity). The presence of the disease was identified by microscopic and visual observation of symptoms and signs of B. cinerea on infected tissues (Hildebrand et al. 2001). Disease incidence was determined as the proportion of floral buds with visual symptoms of Botrytis blossom blight within a stem expressed as a percentage. Disease severity was assessed as the percentage of floral tissue area infected with visual symptoms of Botrytis blossom blight on a stem. A 0–7 disease severity rating scale was used where 0 = no symptoms, healthy plants; 1 = 0–5% affected flower area; 2 = 5–15% affected flower area; 3 = 15–35% affected flower area; 4 = 35–65% affected flower area; 5 = 65–85% affected flower area; 6 = 85–95% affected flower area; 7 = 95–100% affected flower area (Smith 1998; Abbey et al. 2020). Physical development parameters that were recorded included stem length and number of vegetative and floral buds per stem.

Yield components and berry yield

Fifteen stems were selected on 22nd and 28th of July in 2016 and 2020 respectively to assess yield components (set fruit and pinhead/unmarketable berries), where set fruit represents berries that will produce a harvestable fruit and pinhead/unmarketable berries represent deformed berries or fruits which have larger crowns than berries.

Blueberries were harvested on the 12th and 11th of August, 2016 and 2020, respectively with a 40 tine, wild blueberry hand rake from four randomly selected 1 m² quadrants in each plot and the harvest weights were recorded using an Avery Mettler PE 6000 digital balance.

Statistical analysis

Data collected on disease development, physical development parameters, yield components and harvested berries were checked for normality and constant variance on the residuals and data that were not normally distributed were transformed. Harvested berries were cubic-root transformed to ensure data were normally distributed. All the data were analyzed using the PROC GLIMMIX procedure of SAS (version 9.4, SAS institute, Inc., Cary, NC). A combined analysis of data for both seasons was carried out which indicated a significant difference (p < 0.05) between the data from the two seasons, hence the seasons were analyzed individually. The fixed effect was treatments, and replication was the random effect. Least Significance Differences (LSD) was used for multiple means comparison at α = 0.05 when the P-value in ANOVA indicates a significant difference (P < 0.05) among the treatment means.

Results

In the first experimental season after thermal treatment and calcium polysulphide applications, the incidence of Botrytis stem canker before bloom ranged from 0 to 44.5% (Table 1). Treatment combinations that involved thermal treatment had at least a 90% reduction in Botrytis stem canker incidence compared to the untreated control. There was, however, no significant treatment effect of stand-alone application of calcium polysulphide, T. harzianum and the combination of calcium polysulphide + T. harzianum on Botrytis blossom blight incidence compared to the untreated control (Table 1). Generally, plots that had the thermal treatment had lower B. cinerea incidence and severity on stems. Botrytis blossom blight incidence and severity at mid-bloom ranged from 0 to 3.35% and 0 to 3.37%, respectively. At mid-bloom, disease incidence was significantly reduced with the thermal treatment + calcium polysulphide + T. harzianum combinations as well as stand-alone application of thermal treatment, calcium polysulphide and T. harzianum (Table 1). Compared to the untreated control, all the treatments reduced disease incidence with over 50% less incidence at the pre-bloom, mid-bloom and full-bloom stage. Thermal treatment + calcium polysulphide, thermal treatment + T. harzianum, thermal treatment, calcium polysulphide + T. harzianum and calcium polysulphide reduced disease development with over 74% less severity compared to the untreated control (Table 1). At full bloom, disease incidence and severity ranged from 2.27 to 13.2% and 2.87 to 9.30%, respectively. Thermal treatment + calcium polysulphide + T. harzianum, thermal treatment + calcium polysulphide, and calcium polysulphide + T. harzianum provided the most disease suppression with 74.9, 82.8 and 81.8% less incidence. Thermal treatment + T. harzianum and stand-alone thermal treatment, calcium polysulphide and T. harzianum application resulted in reduced disease development compared to the untreated control with 43.0, 52.2, 38.6 and 54.6% less incidence. All the treatments and treatment combinations resulted in over 40% less severity compared to the untreated control (Table 1).

Table 1. Effect of thermal treatment, calcium polysulphide and Trichoderma harzianum T-22 treatment single or combine on incidence and severity of Botrytis blight observed from a commercial wild blueberry field at Debert, Nova Scotia in 2016

	Pre-bloom	Mid-bloom		Full-bloom
Treatment	Botrytis stem canker incidence (%)	Incidence (%)	Severity (%)	Incidence (%)	Severity (%)
Control	39.5a	3.35a	3.37a	13.1a	9.30a
Thermal treatment + Calcium polysulphide + T. harzianum	0b	1.66b	1.89ab	3.31 c	3.62b
Thermal treatment + Calcium polysulphide	0b	0b	0b	2.27 c	2.87b
Thermal treatment + T. harzianum	0b	0.59b	0.62b	7.51b	4.45b
Thermal treatment	2.00b	0.56b	0.87b	6.30bc	4.10b
Calcium polysulphide + T. harzianum	34.2a	0b	0b	2.40 c	2.95b
Calcium polysulphide	38.0a	0.56b	0.72b	8.09b	5.65b
T. harzianum	44.5a	1.00b	1.89ab	5.98bc	5.32b
P-value	0.0001	0.0014	0.010	0.0001	0.0033

Thermal treatment was done on 22nd and 2nd of May 2015 and 2019. Lime sulphur was applied on 25th and 21st of November 2015 and 2019 respectively. Trianum was applied on 26th May 2016 and 4th June, 2020 respectively. Disease assessments were done on 9th May 2016 for pre-bloom, 1st June 2016 for mid-bloom and 15th June 2016 for full-bloom. Mean separation was completed using the LSD test procedure. Data in a column with the same letters are not significantly different at α = 0.05.

In the second experimental season incidence of Botrytis stem canker ranged from 9.3 to 72.0% among the treatments (Table 2). All treatment combinations with thermal treatment, resulted in at least 77% less incidence compared to the untreated control (Table 2). On the contrary, there was no significant treatment effect of stand-alone calcium polysulphide, T. harzianum, and calcium polysulphide + T. harzianum combination on canker incidence compared to the untreated control (Table 2). Disease incidence and severity at mid-bloom ranged from 0 to 4.14 and 0 to 0.82%, respectively. All the treatments reduced disease development with over 72 and 79% less incidence and severity, respectively, compared with the untreated control. There was however no significant difference among the treatments and treatment combinations (Table 2). At full bloom, disease incidence and severity ranged from 0 to 4.47 and 0 to 0.61%, respectively. Similar to the disease reduction pattern at mid-bloom, all the treatment and treatment combinations significantly reduced disease development with more than 77 and 75% less incidence and severity, respectively compared to the untreated control (Table 2).

Table 2. Effect of thermal treatment, calcium polysulphide and Trichoderma harzianum T-22 treatment single or combine on incidence and severity of Botrytis blight observed from a commercial wild blueberry field at Debert, Nova Scotia in 2020

	Pre-bloom	Mid-bloom		Full-bloom
Treatment	Botrytis stem canker incidence (%)	Incidence (%)	Severity (%)	Incidence (%)	Severity (%)
Control	59.3ab	4.14a	0.82a	4.47a	0.61a
Thermal treatment + Calcium polysulphide + T. harzianum	13.3 c	0.33b	0.01b	0.44b	0.07b
Thermal treatment + Calcium polysulphide	9.3 c	1.14b	0.17b	0b	0b
Thermal treatment + T. harzianum	12.0 c	0b	0b	0.67b	0.13b
Thermal treatment	12.0 c	0.41b	0.07b	1.00b	0.15b
Calcium polysulphide + T. harzianum	72.0a	0b	0b	0.89b	0.15b
Calcium polysulphide	49.3b	0.87b	0.15b	0.27b	0.07b
T. harzianum	54.2ab	0b	0b	0b	0b
P-value	0.0001	0.0001	0.0001	0.0001	0.0001

Disease assessments were done on 22nd May 2020 for pre-bloom, 10th June 2020 for mid-bloom and 18th June 2020 for full-bloom. Mean separation was completed using the LSD test procedure. Data in a column with the same letters are not significantly different at α = 0.05.

In the first season, significant treatment effects on average (pre-bloom, mid-bloom and full-bloom) stem length and floral bud and vegetative bud numbers were observed (Table 3). Stems from thermal treatment + calcium polysulphide were taller (20.4 cm) than stems from the untreated control (19.6 cm). On the contrary, stems from calcium polysulphide + T. harzianum and stand-alone T. harzianum were shorter than the untreated control. Similarly, thermal treatment + calcium polysulphide treated plots had had significantly a higher number of floral buds compared with the control. Thermal treatment + calcium polysulphide + T. harzianum and stand-alone thermal treatment had the highest number of vegetative buds compared with the untreated control (Table 3). With regards to yield component, no significant treatment effect was observed on set fruit per stem. On the contrary, thermal treatment + calcium polysulphide, thermal treatment + T. harzianum, calcium polysulphide + T. harzianum and stand-alone calcium polysulphide had more pinheads than the untreated control. Regarding harvestable berry yield, although there was a significant treatment effect, none of the treatments were significantly different from the untreated control (Table 3). Nonetheless, thermal treatment + calcium polysulphide, thermal treatment + T. harzianum, thermal treatment and T. harzianum were the treatments with higher (> 710 g m⁻²) berry yields whereas calcium polysulphide had the lowest berry yield (590.9 g m⁻²) (Table 3).

Table 3. Effect of thermal treatment, calcium polysulphide and Trichoderma harzianum T-22 treatment single or combine on plant growth, yield component and yield harvestable berry harvestable berry yield of wild blueberries from a commercial field at Debert, Nova Scotia in 2016

Treatment	Stem length (cm)	Floral bud number	Vegetative bud number	Set Fruit	Pin heads	Harvestable berry yield (g m^−2)
Control	19.6bc	4.87bcd	9.68 cd	13.2	0.78 c	696.4abc
Thermal treatment + Calcium polysulphide + T. harzianum	20.0ab	4.97bc	10.8a	13.5	1.27abc	681.2abc
Thermal treatment + Calcium polysulphide	20.4a	5.95a	10.2abc	13.2	1.41ab	783.8a
Thermal treatment + T. harzianum	19.3bcd	4.63 cde	9.77 cd	15.8	1.33ab	759.1a
Thermal treatment	19.6bc	4.41e	10.5ab	14.7	1.12bc	710.0ab
Calcium polysulphide + T. harzianum	18.7d	4.54de	9.41d	16.3	1.73a	640.7bc
Calcium polysulphide	19.2 cd	4.79bcde	9.75 cd	13.8	0.89b	590.9 c
T. harzianum	18.7d	5.10b	9.84bcd	14.6	0.77 c	774.5a
P-value	0.0001	0.0001	0.0019	0.0869	0.0035	0.0068

Yield component assessments were done on 22nd July 2016 and harvesting was done on 11th August 2016. Mean separation was completed using the LSD test procedure. Data in a column with the same letters are not significantly different at α = 0.05.

In the second season, significant treatment effects on stem length, and vegetative buds were observed (Table 4). Stems from all thermal treatment plots and thermal treatment combinations with calcium polysulphide and T. harzianum were taller than stems from the untreated control and treatments (Table 4). Plots that received thermal treatment + calcium polysulphide had the highest number of vegetative buds among all the treatments (Table 4). Regarding yield components, plots that received thermal treatment as well as stand-alone T. harzianum had higher fruit set than the untreated control (Table 4). Similarly, to set fruit, the number of fruit pinheads was higher in thermal treated plants compared to the untreated control, except the stand-alone thermal treatment. Although berry yields were significant in the 2020 season, most of the treatments were not different from the untreated control (Table 4). It is, however, important to note that thermal treatment + calcium polysulphide + T. harzianum had the highest berry yield (995.1 g m⁻²). Stand-alone T. harzianum had lower yield than all other treatments except for calcium polysulphide + T. harzianum.

Table 4. Effect of thermal treatment, calcium polysulphide and Trichoderma harzianum T-22 treatment single or combine on plant growth, yield component and yield harvestable berry harvestable berry yield of wild blueberries from a commercial field at Debert, Nova Scotia in 2020

Treatment	Stem length (cm)	Floral bud number	Vegetative bud number	Set Fruit	Pin heads	Harvestable berry yield (g m^−2)
Control	17.6de	6.65	12.9bc	16.8d	1.33de	949.9ab
Thermal treatment + Calcium polysulphide + T. harzianum	19.7a	6.60	13.3bc	25.9a	2.55ab	995.1a
Thermal treatment + Calcium polysulphide	19.8a	7.04	16.4a	24.2ab	2.79a	905.3ab
Thermal treatment + T. harzianum	18.2bc	6.98	13.9b	21.4bc	2.32abc	945.0ab
Thermal treatment	18.7b	6.75	13.1bc	21.0bc	1.76cde	950.1ab
Calcium polysulphide + T. harzianum	17.2e	6.15	12.6bc	18.1 cd	1.08e	814.7bc
Calcium polysulphide	18.0 cd	6.18	12.2 c	20.7bcd	1.28de	930.8ab
T. harzianum	18.2bcd	6.02	12.3c	20.9bc	1.87bcd	733.9 c
P-value	0.0001	0.0688	0.0001	0.0003	0.0001	0.0217

Yield component assessments were done on 28th July 2020 and harvesting was done on 12th August 2020. Mean separation was completed using the LSD test procedure. Data in a column with the same letters are not significantly different at α = 0.05.

Discussion

In this study, we investigated the individual and combined effects of thermal treatment, calcium polysulphide and T. harzianum T-22 against Botrytis blossom blight development in wild blueberry. Results from this study demonstrated that single application of thermal treatment, calcium polysulphide and T. harzianum and their combinations were consistently effective in reducing Botrytis blossom blight development in blueberry.

The development of Botrytis blossom blight is highly dependent on environmental conditions (temperature, rainfall, and leaf wetness) (Delbridge and Hildebrand 2007), therefore, the difference in disease development between 2016 and 2020 can be attributed to the difference in weather conditions. In 2016, there were many occurrences of weather conditions conducive for B. cinerea infection (six infection periods) during bloom, whereas in 2020, there were only three periods conducive for infection (Supplemental material: Figs S-1, S-2, Tables S-1, S-2). This difference in infection period occurrence between the years likely explains why disease incidence and severity were higher in 2016 compared with 2020 (Tables 3.1b-c, 3.2b-c).

In wild blueberry fields, disease development at mid-bloom is generally lower than what was observed at full bloom in the 2016 season. Blueberry flowers are more susceptible to B. cinerea infection when the corolla is fully opened (anthesis) (Hildebrand et al. 2001; Abbey et al. 2018). As flowers advance in their development and reach anthesis, more delicate tissues such as the corolla, filament/anther and style are exposed to pathogens. These floral tissues are less waxed, lack lignified dermal tissues and mostly have very thin cuticles that do not create sufficient barrier to limit pathogen infection (Lacaze and Joly 2020). Furthermore, an increase in membrane permeability as well as increased pollen and pollen exudates provide pathogens with an easy pathway and source of nutrients and moisture for infection to occur at anthesis (Fourie and Holz 1998). At mid-bloom, most of the floral tissue were at/below the more resistant floral stages, F5 (corolla half-developed) whereas at full bloom, most of the flowers were at the most susceptible stage, F7 (fully open).

In this study, treatment combinations that involved thermal treatment were effective in reducing canker development on the stems before bloom. This could be due to thermal treatment at the beginning of the sprout year, inactivating or killing the initial B. cinerea inoculum on field debris. Given this, it is possible that stems from these thermal plots were exposed to less inoculum, and hence fewer stem cankers developed.

At the onset of this trial, we postulated that thermal treatment would help reduce the development of Botrytis blossom blight. This was based on observations and previous knowledge that thermal treatment has the advantage of reducing Botrytis blossom blight in wild blueberry fields (Delbridge and Hildebrand 1997; Yarborough 2004). Therefore, it is not surprising that thermal treatment yielded a substantial reduction in Botrytis blight development. The reduction in disease by thermal treatment may be due to the additive effect of debris destruction as the pathogen is deprived of substrate and there is a reduction in the initial inoculum load that causes primary infections.

Application of T. harzianum T-22 and its combination with thermal treatment positively reflected on plant disease suppression and plant development (stem length) as well as yield component (set fruit) in the second year (Table 4). These results agree with the reports of many studies that have employed Trichoderma spp. as single application or in combination with other treatments (Elad 2000; Ranasingh et al. 2006; Elad and Stewart 2007; McGourty et al. 2011; Vos et al. 2015). In a greenhouse study, the application (3 doses) of Trichodex (T. harzianum T-39) at 4 kg ha⁻¹ reduced Botrytis grey mould on tomato by over 50% compared with an untreated control and more than 70% compared with chemical control (Fiume and Fiume 2006). In another study, Barakat and Al-Masri (2017) reported up to a 45% reduction in disease severity of grey mould on strawberry leaves with the application of T. harzianum (Jn14). Finally, Freeman et al. (2004) demonstrated the ability of the T. harzianum T-39, T-161 and T-166 isolates to control B. cinerea in strawberry. In their study, isolate T-39 applied at 2 day intervals, T-166 applied at 7 day intervals, or T-161 combined with T-39 at 7 day intervals, were as effective as fenhexamide, a chemical fungicide. Although effective, the disease control abilities of BCAs are known to show some level of inconsistency under field conditions. In view of this, different combination of BCAs or their combination with other disease control strategies is highly encouraged. Therefore, the ability of the combination of T. harzianum and its combination with calcium polysulphide and thermal treatment to reduce disease control in both years is important and confirms the disease control ability of T. harzianum.

Calcium polysulphide has been reported to kill B. cinerea sclerotia and significantly reduce inoculum, hence making it a good clean-up product (Bettiga 2013). As a caustic material, calcium polysulphide can burn and destroy pathogens that overwinter in the field. Due to its effectiveness, calcium polysulphide has been used to control anthracnose (Elsinoe veneta) and spur blight (Didymella applanata) in brambles, black rot (Guignardia bidwelli) and powdery mildew (Uncinula necator) in grapes (Longstroth 2011), and Exobasidium leaf and fruit disease in blueberries (Brannen et al. 2016). The ability of calcium polysulphide to reduce Botrytis blossom blight in this study confirms the outcome of these previous studies. Given the caustic nature of calcium polysulphide, it was applied during the dormant stage of wild blueberry plants to avoid plant tissue damage. It is important to note that the time of application of the factors in this study ensured that at every phase of the 2-year production cycle, there was a Botrytis blossom blight management component on the field. It is therefore not surprising that consistent disease reductions were achieved in both years with the application and combination of thermal treatment, calcium polysulphide and T. harzianum.

Although the various stand-alone treatments and their combinations resulted in similar disease control levels, the combination of thermal treatment, calcium polysulphide and T. harzianum is most preferable. The combination of these treatments with different modes of action and time of application falls in line with the concept of integrated pest management. This approach will protect the different components of the management strategy from total failure. For instance, the application of T. harzianum will help prevent total disease control failure in the event that the environmental conditions or disease outbreak do not favour just a single treatment. This is important because B. cinerea is a polycyclic pathogen whose outbreak relies heavily on environmental conditions.

Over the years, an annual wild blueberry loss between 20 to 35% due to Botrytis blight have been reported (Delbridge and Hildebrand 1997; Abbey et al. 2020). Given this, the significant disease reduction (> 40%) obtained with the treatment combinations in this study is very important, and similar disease control was obtained with commercial fungicides in previous studies (Abbey et al. 2020, 2021). It is worth noting that these disease management approaches can be applied before the B. cinerea infection window, yet they offer significant benefits with respect to lowering the primary inoculum sources. They also offer the advantage of reducing the risk of resistance development to conventional fungicides due to reduced usage of chemical fungicides. The suppression of B. cinerea infection by thermal treatment of plant debris, application of calcium polysulphide and biofungicides is useful in maintaining the perception of wild blueberries as a low input commodity. The outcome of this study could also have a practical implication for fields with limited fungicide usage, organic wild blueberry production or fields with widespread fungicide resistance in the B. cinerea population. Given that there was no application of any chemical during the cropping year, berries produced from this study are free from any chemical residue. With the majority of wild blueberries being exported to Europe and Asia where there are strict MRLs, it is important to develop disease control measures that will result in residue-free berries.

In addition to Botrytis blight, other diseases of wild blueberry occur. These include rust (Naohidemyces vaccinia), septoria leaf spot (Septoria spp.) and red leaf (Exobasidium vacinii), which infect stems and leaves, as well as others such as anthracnose (ripe rot) (Colletotrichum spp.) and Monilinia blight (Monilinia vaccinii-corymbosi) that infect stems, flowers, and fruits (Percival 2013; AAFC 2017). The effect of these diseases on wild blueberry includes leaf drop, loss of leaf area, increased premature berry drop and reduction in berry yield. Given their importance and their regular presence on commercial wild blueberry fields, disease management programs that have the potential to contribute to a reduction in the impact of these diseases is very important. Although these other diseases were not assessed in this study, the treatments and their combinations evaluated in this report have the potential to alleviate the effect of these diseases by destroying pathogen inoculum as has been reported in previous studies (DeGomez 19881988; Jamar et al. 2017; DeLong et al. 2018).

This study revealed a significant treatment effect on physical development of blueberry plants. Generally, plots that received thermal treatment or thermal treatment combined with calcium polysulphide and T. harzianum T-22 had taller stems and higher numbers of floral and vegetative buds. This outcome is consistent with and supports the observation that the number of sprouts, length of stems, and the total number of flower buds per stem were greater when thermal treatment was done in early spring (Eaton and White 1960). This plant development features can be attributed to the thermal treatments which facilitate the release of inorganic nutrients that can be used by the plant for their development.

Disease control obtained with thermal treatment and its combination with calcium polysulphide did not translate into an increase in berry yield. Interestingly, T. harzianum had lower yield than all the treatments in the second year but higher yield in the first year. The similarities and inconsistency in the yield among the various treatments may partly be attributed to the dry weather conditions experienced from the mid to late stages of the growing seasons and the enormous variability in wild blueberry fields. Wild blueberry fields are characterized by excessive plant to plant variability. The variability is so extreme that it is almost impossible to find two morphologically identical clones in the same field (Kinsman 1993). This variability, coupled with the irregularity of plant density throughout the fields and the mixture of different phenotypes and species with different yield potentials, makes it very challenging to establish conclusions based on only yield. Given that this study was conducted over two seasons of 2-year production cycles, it would be informative to see the effects of this approach over a longer term. This will potentially generate enough and consistent data to draw firm conclusions, given the extreme variabilities in wild blueberry fields.

In conclusion, the results from this study provide evidence that field sanitation and other practices that reduce pathogen inoculum and field debris are potential integrated disease management strategies that can be adopted to reduce Botrytis blight symptoms in wild blueberry fields. The temporal application and combination of thermal treatment, calcium polysulphide and biofungicide provide a useful management strategy that takes into consideration the timing of pathogen and plant development and the susceptible floral development stages during the 2-year production cycle. The outcome of this study indicates that the combination of field sanitation, calcium polysulphide and biofungicides has the potential to reduce Botrytis blossom blight in wild blueberry fields. This information can help to reduce chemical fungicide usage and the total dependency on only chemical fungicides during the cropping year.

Acknowledgements

We gratefully thank the Bragg Lumber Company, the Wild Blueberry Producers Association of Nova Scotia, and all research assistants and interns for their contributions. The mention of a product or trade name does not constitute a guarantee or warrantee of the product by Dalhousie University nor an endorsement over similar products mentioned.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online here: https://doi.org/10.1080/07060661.2021.2023649

Additional information

Funding

The authors are grateful to the Nova Scotia Department of Agriculture Research Acceleration Program for financial support.