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  table top indoor production

 

USDA-ARS, Appalachian Fruit Research Station, Kearneysville, WV

One of the biggest challenges in strawberry production in the United States is managing diseases and pests. Diseases such as gray mold (caused by Botrytis cinerea), anthracnose (caused by Colletotrichum acutatum), or powdery mildew (caused by Podosphaera aphanis) can cause severe losses by reducing fruit quality and yield as well as causing fruit decay during production and after harvest, if not controlled beginning early on in the production cycle (Burlakoti et al., 2013; Carisse et al., 2013; Smith, 2013; Xiao et al., 2001). For control of gray mold and powdery mildew, it has been paramount that the control measures begin in the field at bloom, to protect flowers from infections (Figs. 1 and 2) that, in the case of gray mold, may account for up to 80 percent of fruit decay (Bulger et al., 1987).

 

Healthy (left), Botrytis cinerea (center) and Podosphaera aphanis-infected (right) strawberry flowers.

Fig. 1. Healthy (left), Botrytis cinerea (center) and Podosphaera aphanis-infected (right) strawberry flowers.

Fungicides traditionally have been used for controlling these diseases with regular applications from the early flowering stage through harvest (Bulger et al., 1987; Mertely et al., 2002; Wedge et al., 2007; Wilcox and Seem, 1994). However, their use has increasing limitations due to rapidly developing resistance to commonly used fungicides, new regulations limiting use of pesticides, especially in protective cultures, and growing demand for fruit free of pesticide residues (Wedge et al., 2007; Pokorny et al., 2016; Smith, 2013).

 

Development of gray mold from Botrytis cinerea infected petals.

Fig. 2. Development of gray mold from Botrytis cinerea infected petals.

Biological approaches using various beneficial fungi and bacteria to control strawberry diseases have been explored with considerable success, but they are not as effective as conventional fungicide treatments. Despite positive attitudes by growers toward this approach, commercial biocontrol products have been used rarely in strawberry production (Moser et al., 2008). However, biological control can be combined with compatible physical or chemical treatments to increase disease control, as it was clearly demonstrated in many examples on various fruit after harvest (Janisiewicz and Conway, 2011).

UV irradiation has been used to kill microorganisms in various systems including the sterilization of air in hospitals, water in treatment plants, and to some extent in the food industry (Beggs et al., 2006; Bintsis et al., 2000; Gardner and Shama, 2000). Use of UV in crop protection has been sporadic and mostly exploratory. The main reasons for this has been the damaging effect (e.g. leaf burn and fruit discoloration and softening) of UV irradiation to plants at the doses required to kill pathogens, the impracticability of long exposure time (several minutes) of irradiation with UV-B (less powerful than UV-C) used in most instances, and the associated energy cost. For example, treatment of harvested strawberries with UV-C alone, or with a combination of pulsed white light and heat, significantly reduced fruit decay in storage; however, the reductions were still below commercially acceptable levels (Marquenie et al., 2003; Nigro et al., 2000; Pombo et al., 2011; van Delm et al., 2014).

Recently, Janisiewicz et al. (2015, 2016a,b) showed that using UV-C irradiation at night kills strawberry pathogens causing gray mold, anthracnose and powdery mildew at much lower doses than daytime irradiation. The dark period following irradiation at night most likely prevented activation of a light-induced DNA repair mechanism in microbes after their DNA was damaged by UV-C irradiation (Beggs, 2002) and increased the killing power of UV-C by 6 to 10-fold, depending on the pathogen. This allowed for use of reduced UV-C doses that were effective in killing pathogens without damaging leaves, flowers, or fruit. This treatment was also effective in reducing mite infestations below accepted treatment threshold levels (Short et al., 2018).

 

The inclusion of a four hour dark period resulted in complete killing of C. acutatum (Fig. 3) and almost complete killing of B. cinerea conidia in a laboratory plate assay on agar medium at a dose of 12.36 J/m2 (60 sec exposure) (Janisiewicz et al., 2016 a, b).

 

Formation of Colletotrichum acutatum colonies from conidia irradiated with UV-C for various times (0 to 60 seconds) and exposed to daylight either immediately after irradiation (group on the left) or after four hours incubation in dark (group on the right). Photograph taken after incubating plates at room temperature for 72 hours.

Fig. 3. Formation of Colletotrichum acutatum colonies from conidia irradiated with UV-C for various times (0 to 60 seconds) and exposed to daylight either immediately after irradiation (group on the left) or after four hours incubation in dark (group on the right). Photograph taken after incubating plates at room temperature for 72 hours.

The powdery mildew fungi reside mainly on plant surfaces and are vulnerable to UV treatment. In earlier work, powdery mildew on roses was reduced by irradiation with UV-B; however, with this wavelength, the main effect was physiological resulting in a reduction in sporulation and treatments of more than five minutes were needed to achieve any significant effect, making this approach impractical (Suthaparan et al., 2010). To test the effect of our UV-C/dark treatment on powdery mildew of strawberries, we developed a leaf disc assay where we cut leaf discs from strawberry leaves at the most susceptible early stage of development and used the underside of the leaf for brush-inoculation with conidia of the fungus collected from leaves of powdery mildew-infected strawberry plants. The discs were then placed on water agar medium with streptomycin to prevent bacterial growth and were irradiated with UV-C, incubated in the dark for four hour, and then incubated at 14-21 °C (night/day temperature) for three days and for additional eight days at 22 °C with ~ 10 h light/day. The first powdery mildew symptoms were visible three days post inoculation and the data were collected eleven days post inoculation (Fig. 4). The UV-C/dark treatment significantly reduced the incidence of powdery mildew on discs and the disease occurred only sporadically compared to the non-irradiated control treatment.

 

Strawberry leaf disc assay showing growth of powdery mildew on control disc (left) (left) but not on UV-C/dark treated disc (right).

Fig. 4. Strawberry leaf disc assay showing growth of powdery mildew on control disc (left) but not on UV-C/dark treated disc (right).

In further studies with powdery mildew, entire ‘Monterey’ strawberry plants were inoculated with the fungus and the disease was allowed to develop under greenhouse conditions. After the onset of disease signs, the UV-C/dark treatment was applied once per week for three weeks after which the plants were evaluated for severity of powdery mildew and harvested fruit for yield of diseased and healthy fruit. Visible signs of powdery mildew on strawberry plants treated with UV-C/dark were drastically reduced and were seldom visible on the upper side of the leaves, while on control plants the signs were more apparent (Fig. 5).

 Powdery mildew development on ‘Monterey’ strawberry plants three weeks after the initiation of the UV-C treatment (60 sec irradiation followed by four hour dark period, once per week).

Powdery mildew development on ‘Monterey’ strawberry plants three weeks after the initiation of the UV-C treatment (60 sec irradiation followed by four hour dark period, once per week).

Fig. 5. Powdery mildew development on ‘Monterey’ strawberry plants three weeks after the initiation of the UV-C treatment (60 sec irradiation followed by four hour dark period, once per week). (A) In the upper left corner inset, one of the three leaflets of a strawberry leaf treated with UV-C showing no signs of powdery mildew on the upper surface and signs on the lower surface. (B) Reduction of powdery mildew damage to strawberries by UV-C treatment.

The challenge in controlling powdery mildew is to reach the underside of the leaves with UV-C as this disease develops on both sides of the leaf. The initial experiments were conducted with application of the UV-C from above. We addressed this problem later in large scale experiments by adding reflecting surfaces in high tunnel production and designing a multidirectional UV-C irradiation prototype for table-top production.

The average yield of healthy fruit per UV-C/dark treated plant was significantly higher and diseased fruit significantly lower than on untreated plants (Table 1). In addition, fruit from plants treated with UV-C/dark were larger, had better color and luster, and did not have the cracking that was prominent on non-irradiated fruit (Fig. 5).

Table 1. Fruit yield and quality from powdery mildew-infected ‘Monterey’ strawberry plants treated or not treated with UV-C/dark.
  Treatment Mean fruit weight (g)/plant
Diseased Healthy % Healthy
Control No UV-C 54.50aA 90.24 B 67.4  ±11.1b
UV-C/dark treated  5.56 B    179.40 A               97.0  ±2.0

a Fruit weight means with different letters in columns are different according to t test (P = 0.05).

bStandard error of the mean of five replicates.

UV-C treatment (60 sec followed by a four hour dark period) of harvested, ripe strawberry fruit artificially inoculated with B. cinerea conidia, significantly reduced decay on the fruit after five (Fig. 6) and seven days incubation at room temperature. After seven days, the amount of decay was reduced by 50 percent. Increasing UV-C doses to 90 and 120 sec completely eliminated decay without any negative effect on the appearance of the fruit and sepals.

 

Control of gray mold on strawberries inoculated with B. cinerea using UV-C irradiation for 60 sec followed by four hour dark period and incubation for five days at room temperature.

Fig. 6. Control of gray mold on strawberries inoculated with B. cinerea using UV-C irradiation for 60 sec followed by four hour dark period and incubation for five days at room temperature.

For large scale application, we developed a fully automated self-propelled irradiation apparatus with timers for duration and initiation of the night irradiation for tunnels, and a multidirectional UV-C irradiation apparatus for table top production (Fig. 7).

Prototypes for UV-C irradiation of strawberry plants at night in raised bed high tunnel table top indoor production

Fig. 7. Prototypes for UV-C irradiation of strawberry plants at night in raised bed high tunnel (left) and table top indoor production (right).

To increase robustness of disease control and to assure microbial safety of the UV-C/dark treatment we combined this treatment with application of microbial antagonists. This disease control approach, now called PhylloLux technology, includes spray application of two mutually compatible yeasts, Metschnikowia pulcherrima and Aureobasidium pullulans, following UV-C/dark treatment. In addition to being excellent colonizers of flowers (Fig. 8) and leaves, these yeasts very efficiently fill in the microbial void left after UV-C/dark “sterilization“ which prevents potential recolonization of plant surfaces by unwanted pathogenic microorganisms (Janisiewicz et al., 2017). Both yeasts were originally isolated from fruits, are part of the natural fruit microflora, and have strong biocontrol activity against pathogens causing various fruit decays. In an assay on strawberry petals where all petals in control treatment were infected, M. pulcherrima reduced incidence of infection to 25 percent and A. pullulans to 50 percent seven days after inoculation.

etals inoculated with B. cinerea alone (dishes on the left), B. cinerea and M. pulcherrima (center), and B. cinerea and A. pullulans (right). Fig. 8. Populations of two yeast antagonists, M. pulcherrima and A. pullulans, on detached strawberry flowers and anthers at various times after spray application (chart on left), and their effect on B.etals inoculated with B. cinerea alone (dishes on the left), B. cinerea and M. pulcherrima (center), and B. cinerea and A. pullulans (right).

Fig. 8. Populations of two yeast antagonists, M. pulcherrima and A. pullulans, on detached strawberry flowers and anthers at various times after spray application (chart on left), and their effect on B. cinerea infection in a petal assay four and seven days after inoculation (picture on right). Petals inoculated with B. cinerea alone (dishes on the left), B. cinerea and M. pulcherrima (center), and B. cinerea and A. pullulans (right).

The other benefits of the PhylloLux technology are its ability to promote fruit production in short-day cultivars (Takeda et al., 2018) and to control mites and insects. Two-spotted spider mite (Tetranychus urticae) is the major pest feeding on strawberry plants (Fig. 9) which causes losses resulting in reduced photosynthetic activity, lower fruit weight, yield reduction and damaged fruit.

Two-spotted spider mite and eggs on underside of strawberry leaf.

Fig. 9. Two-spotted spider mite and eggs on underside of strawberry leaf.

This mite is managed mainly by application of acaricides; however, similarly to the situation with disease control, they have developed resistance to most of the pesticides (Van Leeuwen et al., 2010). Applications of predatory mites as biological control agents can also be used as a control technique, but they can be susceptible to both broad spectrum pesticides as well as acaricides targeting pestiferous mite species (Amoah et al., 2016).

Our original observation of a great reduction in mite infestation on potted strawberry plants treated with UV-C/dark twice a week was confirmed in very controlled studies with artificial infestation with two-spotted spider mites and nightly exposure to UV-C for 60 second for four weeks in a Phytotron greenhouse. The UV-C irradiation treatment dramatically reduced mite populations below the accepted economic threshold of five mites per mid-canopy leaflet compared to nearly 200 mites per mid-canopy leaflet on untreated plants. None of the UV-C irradiated strawberry plants had any spider mite webbing; whereas, >50 percent of untreated plants were webbed. In a subsequent experiment where artificially infested plants were allowed to develop very high mite infestation (~500 mites/leaflet) before the first UV-C/dark treatment, the infestation was reduced to acceptable economical level on UV-C/dark treated plants, while control plants were totally devastated at the end of the four week experiment (Fig. 10).

Two-spotted spider mite infestation on UV-C/dark treated and untreated (Control) strawberry plants. Populations of mites were allowed to build up to a high level before beginning the UV-C/dark treatment.

Fig. 10. Two-spotted spider mite infestation on UV-C/dark treated and untreated (Control) strawberry plants. Populations of mites were allowed to build up to a high level before beginning the UV-C/dark treatment.

Summary

A search for alternatives to synthetic fungicides for control of strawberry diseases has led us to the development of PhylloLux technology that combines UV-C irradiation followed by a specific dark period (two-four hour depending on pathogen) and application of microbial antagonists. This technology is safe and should be compatible with organic fruit production, which further increases its value. In addition to controlling plant pathogens, it also controls some arthropods and promotes early fruit production in short-day cultivars. The potential of this technology goes well beyond its application to strawberries and may include applications in production of other fruit and vegetable crops as well as ornamental plants and nursery stocks. As with any new technology, its future lies in the hands of its developers and users, and at present it appears that the question is not if, but how soon it will be broadly implemented.

For full list of complete citations please contact Wojciech J. Janisiewicz at: This email address is being protected from spambots. You need JavaScript enabled to view it.