Improving Biopesticide Efficacy of Apple Diseases through Co-application with Natural Products

Progress report for GNE19-198

Project Type: Graduate Student
Funds awarded in 2019: $14,685.00
Projected End Date: 10/31/2022
Grant Recipient: University of New Hampshire
Region: Northeast
State: New Hampshire
Graduate Student:
Faculty Advisor:
Anissa Poleatewich
University of New Hampshire
Expand All

Project Information

Project Objectives:

The overall goal of this research is to improve biopesticide efficacy in suppressing apple scab through finding synergisms with the natural product, chitosan. Reaching this goal will help farmers to reduce losses, decrease reliance on fungicides, and improve overall yield. The specific objectives are to;

1. Compare commercial chitosan formulations and application rates for efficacy in controlling foliar apple scab

2. Investigate synergisms between chitosan-based treatments and microbial biopesticides for suppression of apple scab in laboratory and on-farm studies.

3. Investigate the potential for chitosan formulations to reduce the quantity and viability of overwintering spores of Venturia inaequalis in orchard leaf litter.

Introduction:

Apple scab, caused by the fungus Venturia inaequalis (Cke.) Wint., is the most destructive disease of apples in the Northeast U.S. where warm, moist conditions can cause severe infections and devastating crop losses to farmers (MacHardy, 1996). Apple scab can cause up to 100% crop loss and significant reduction in fruit marketability due to consumers’ low threshold for imperfection on their apples (Vaillancourt and Hartman, 2000). Apple growers rely on multiple fungicides applications per season to manage apple scab (MacHardy, 1996). The development of fungicide resistance (the ability to survive when exposed to a chemical) by V. inaequalis is of large concern and several classes of fungicides, such as benzimidazole and demethylation inhibitors, have already lost their effectiveness (Holb, 2009; Ma and Michailides, 2005). Thus, alternative practices that can be incorporated with sanitation, fungicides, and other IPM strategies are critical to manage apple scab in New England.

One way that farmers are reducing their use of chemical pesticides is by utilizing naturally occurring compounds that can promote plant health and suppress disease. One strategy is to use beneficial microbes (biopesticides) to suppress disease. Many growers have incorporated biopesticides into their spray programs with mixed success. These inconsistencies in performance remain a barrier to broad adoption. However, biopesticide may be more effective at reducing disease through synergisms with natural compounds. A promising natural compound, chitosan, a deacetylated derivative of chitin, has been documented to have antifungal properties and promote plant growth (Zhang et al., 2011). Other benefits include enhanced photosynthesis, resistance to abiotic and biotic stress, and increased plant growth and yield (Reddy et al., 2000). Chitosan is an approved food additive in the U.S. and has been used effectively as a postharvest application to prevent disease and extend shelf life of perishable fruits and vegetables (Pichyangkura and Chadchawan, 2015; Romanazzi et al., 2018). There are limited examples of research evaluating preharvest application of chitosan on tree fruits. However, success in vegetable crops to promote plant health and suppress disease suggests that chitosan may have broader applications in agriculture (El-ghaouth et al., 2000).

Most fungi, including V. inaequalis, contain cell walls composed of chitin (Jerome, 1965). Chitinase is an enzyme that denatures cell walls, effectively degrading the fungi (Boller, 1993). El-Ghaoth et al. (1994) found that chitosan amended soil controlled Pythium root rot on cucumbers and triggered the production of chitinase and other anti-fungal compounds by the host. An application of chitosan acts as a microbial food source and can stimulate production of chitinase enzymes. Researchers have investigated potential synergisms between beneficial microbes and natural products and the implications for disease management. For example, Kokalis-burelle et al. (Kokalis-burelle et al., 1992) reported a 60% reduction in early leafspot of peanut when plants were treated with Bacillus cereus and chitin. It was hypothesized that the chitin stimulated production of anti-fungal enzymes and helped the beneficial microbe persist long enough to compete with the pathogen. These studies suggest that finding synergisms between biopesticides and chitosan has great potential to improve biopesticide efficacy in suppressing diseases. Although there is significant research on chitosan in the literature documenting its potential (Zhang et al., 2011), research is needed to bring best practices to growers and facilitate adoption. The overall goal of this research was to improve biopesticide efficacy in suppressing apple scab in northeastern orchards through finding synergism with the natural product, chitosan. Reaching this goal will help farmers to reduce losses, decrease reliance on fungicides, and improve overall yields.

Boller, T., 1993. Antimicrobial functions of the plant hydrolases, chitinases and 1,3 glucanases, in: Fritig, B., Legrand, M. (Eds.), Mechanisms of Plant Defense Responses. Kluwer Academic Press, Dordrecht, Netherlands, pp. 391–400.

El-Ghaouth, A., Arul, J., Grenier, J., Benhamou, N., Asselin, A., Belanger, R.R., 1994. Effect of chitosan on cucumber plants: Suppression of Pythium aphanidermatun and induction of defense reactions. Phytopathology 84, 313–320.

El-Ghaouth, A., Smilanick, J.L., Wilson, C.L., 2000. Enhancement of the performance of Candida saitoana by the addition of glycolchitosan for the control of postharvest decay of apple and citrus fruit. Post 19, 103–110.

Holb, I.J., 2009. Fungal Disease Management in Environmentally Friendly Apple Production – A Review. Sustain Agric. Rev. 2, 219–293.

Jerome, M., 1965. The cell wall, in: Ainsworth, G., Sussman, A. (Eds.), The Fungi: An Advanced Treatise. Academic Press, New York, pp. 49–76.

Kokalis-burelle, N., Backman, P.A., Rodriguez-kabana, R., Ploper, L.D., 1992. Potential for Biological Control of Early Leafspot of Peanut Using Bacillus cereus and Chitin as Foliar Amendments ’. Biol. Control 2, 321–328.

Ma, Z., Michailides, T.J., 2005. Advances in understanding molecular mechanisms of fungicide resistance and molecular detection of resistant genotypes in phytopathogenic fungi. Crop Prot. 24, 853–863. https://doi.org/10.1016/j.cropro.2005.01.011

MacHardy, W.E., 1996. Apple Scab: Biology, Epidemilogy and Management. American Phytopathological Society Press, St. Paul, MN.

Pichyangkura, R., Chadchawan, S., 2015. Biostimulant activity of chitosan in horticulture. Sci. Hortic. (Amsterdam). 196, 49–65. https://doi.org/10.1016/j.scienta.2015.09.031

Reddy, M.V.B., Belkacemi, K., Corcuff, R., Arul, J., 2000. Effect of pre-harvest chitosan sprays on post-harvest infection by Botrytis cinerea and quality of strawberry fruit. Postharvest Biol. Technol. 20, 39–51.

Romanazzi, G., Feliziani, E., Sivakumar, D., 2018. Chitosan, a Biopolymer With Triple Action on Postharvest Decay of Fruit and Vegetables: Eliciting, Antimicrobial and Film-Forming Properties. Front. Microbiol. 9, 1–9. https://doi.org/10.3389/fmicb.2018.02745

Vaillancourt, L.J., Hartman, J.R., 2000. Apple scab. Plant Heal. Instr. https://doi.org/10.1094/PHI-I-2000-1005-01

Zhang, H., Li, R., Liu, W., 2011. Effects of Chitin and Its Derivative Chitosan on Postharvest Decay of Fruits: A Review. Int. J. Mol. Sci. 12, 917–934. https://doi.org/10.3390/ijms12020917

Cooperators

Click linked name(s) to expand
  • George Hamilton (Educator and Researcher)
  • Jeremy DeLisle (Educator and Researcher)
  • Dr. Kari Peter (Educator and Researcher)

Research

Materials and methods:

Objective 1: Compare commercial chitosan formulations and application rates for efficacy in controlling foliar apple scab

Growth room experiments are being conducted to test the efficacy of several chitosan formulations and concentrations in controlling V. inaequalis on apple seedlings. Currently, apple seedlings are being tested to evaluate chitosan concentrations for phytotoxicity prior to a larger scale greenhouse study with grafted whips.

For preliminary growth room experiments, ‘Macintosh’ and ‘Golden Delicious’ seeds (extracted from fruit purchased at Hannaford) were surface sterilized with 10% bleach for 5 minutes and rinsed (3x) with sterile distilled water for 2 minutes. The seeds were then stratified in sterile sand at 4°C for 3 months. When germinated, the seedlings were transplanted into Ray Leach cone-tainers filled with peat (ProMix Bx) and placed in a walk-in growth room (22°C, 75% relative humidity, and a 12-hour photoperiod). While these seeds are genetically different due to pollination, this is an inexpensive way to develop methods prior to using grafted apple trees.

Non-commercial stock solutions of low molecular weight chitosan (Sigma Aldrich, St. Louis, MO) were prepared by dissolving 5 g of chitosan in 200 mL of sterile MilliQ water with 5 mL of glacial acetic acid (stirred for 24 h at room temperature). The volume was adjusted to 450 mL with sterile MilliQ water and the pH was adjusted to 5.3 by adding 1 M sodium hydroxide (NaOH). The stock solution of chitosan was adjusted to wanted concentrations using sterile Milli Q water. Glacial acetic acid (1% v/v) with the pH adjusted to 5.3 by adding 1 M NaOH was used as a control. High TideTM was obtained from Tidal Vision and applied at the manufacturer’s rate.

Once the apple plants had produced new, green growth (about six leaves), the chitosan treatments (Table 1) were sprayed onto plants until glisten in a fume hood. The plants were allowed to dry and then returned to the growth room. Plant height, number of nodes per plant, and SPAD measurements (leaf chlorophyll content) were measured prior to the chitosan application and 7 days post application to determine the effect of chitosan concentration on plant growth and photosynthesis.

Table 1. Chitosan treatments applied to apple seedlings in preliminary experiments to optimize chitosan dose.

Treatment

Application

1

Chitosan 1%

2

Chitosan 0.5%

3

Chitosan 0.25%

4

Chitosan 0.01%

5

Acetic Acid 1%

6

Milli Q Control

7

High Tide (0.5%)

 

Next, ‘Macintosh’ seeds (extracted from fruit collected at a local farm) were prepared and grown as previously described in a growth room. Non-commercial stock solutions of low, medium, and high molecular weight chitosan (Sigma Aldrich, St. Louis, MO) were prepared as previously described with the addition of a nonionic surfactant, CapSil (0.4 mL/L), which was included to improve contact with the leaves. Different concentrations (2, 1.5, 1, 0.75, 0.5%) of these chitosan products were applied to the apple seedlings to determine the potential for phytotoxicity. Additionally, three commercial products (Armour-Zen, Tidal Wave 1%, and Tidal wave 2%) were evaluated. Application and data collection will be the same as previously described.

In the next few months, the three reagent grade chitosan formulations and the three commercial products (Table 2) will be applied on apple seedling of cultivar ‘Macintosh’ (rates will be based on the previous experiment) to evaluate disease resistance. Acetic acid and the MilliQ treatments act as controls. This experiment will be set up as a randomized complete block design with 6 replicate plants per treatment. In each treatment, half of the plants will be inoculated with V. inaequalis and half will remain non-inoculated to observe the effects of the chitosan treatments on plant health and growth. The trial will be repeated twice.

Table 2. Chitosan treatment sprays

Treatment

Product

1

Low Molecular

2

Medium Molecular

3

High Molecular

4

Acetic Acid

5

Armour-Zen 15%

6

Tidal Wave 1%

7

Tidal Wave 2%

8

Milli Q + surfactant

9

Milli Q water control

 

The chitosan treatments will be applied as previously discussed. Seven days after the treatment application, the plants will be inoculated with V. inaequalis. An isolate of V. inaequalis was received from Dr. K. Peter at Penn State University’s Fruit Research and Extension Center (FREC). To prepare inoculum, cultures were maintained on ¼ PDA for 2-3 weeks. The isolate was placed in a Filter Blender bag with sterile Milli-Q water and blended into a mycelial slurry. The resulting spore suspension was filtered through cheesecloth and the concentration of the inoculum was adjusted to 3.0 x 104 conidia mL-1 using a hemocytometer. Immediately prior to scab inoculations, one drop of Tween 20 was added to the conidial suspension to minimize spore clumping. The spore suspension will then be sprayed on the plants to run-off. The non-inoculated, control plants will be sprayed with sterile water. The plants will be placed in a humid chamber (18°C and 100% relative humidity) for 48 hours and then returned to the growth chamber. 

At 14 days post inoculum, overall plant disease severity will be measured using a five-point scale rating (0=no disease, 1=20%, 3=60%, 4=80%, and 5=100% or total plant collapse). Four leaves per plant will be evaluated for disease severity of apple scab using WinRHIZOTM (Regent Instruments, Quebec), a software which is an image analysis system which can be used for imaging leaves and calculating disease leaf area. Plant height, number of nodes per plant, and SPAD measurements (leaf chlorophyll content) will be measured for all treatments weekly to determine the effect of chitosan concentration on plant growth and photosynthesis. At completion of the trial, plant dry biomass will be collected. Disease severity and plant health data will be analyzed for statistical significance using a One-Way Analysis of Variance (ANOVA) in JMP Pro 14 (SAS Institute Cary, NC). The model statement will include chitosan treatment as the independent variable, block as the random variable, and disease severity, height, nodes, SPAD, and dry weight as dependent variables. Statistical significance will be assessed at p < 0.05 and a Tukey Honest Significant Difference (HSD) Post-hoc test will be used to separate the means. 

Objective 2: Investigate synergisms between chitosan-based treatments and microbial biopesticides for suppression of apple scab in laboratory and on-farm studies.

Controlled environment evaluations. The chitosan-based treatments and microbial biopesticides will be evaluated on apple plants in a controlled environment growth chamber. This experiment will consist of a 4 x 4 factorial with four chitosan treatments (3 chitosan formulations + water control) and four biopesticide treatments (3 commercial products + water control). Treatments will be arranged in a randomized complete block design with 10 replicate plants per treatment (160 plants total). In each treatment, half of the plants will be inoculated with V. inaequalis and half remained non-inoculated to observe the effects of the treatments on plant health and growth. The trial will be repeated twice.

Apple seedlings will be propagated as discussed in objective 1. Once the plants have three true leaves, the chitosan and biopesticide treatments will be applied. Seven days after the treatment application, the plants will be inoculated with V. inaequalis as discussed in objective 1. At 14 days post inoculum, disease severity will be assessed as discussed in objective 1. To evaluate plant health and photosynthesis for both inoculated and non-inoculated plants, plant height, number of nodes per plant, dried biomass, and SPAD measurements (leaf chlorophyll content) will be measured. Disease severity and plant health data will be analyzed for statistical significance using a Two-Way ANOVA in JMP Pro 14 (SAS Institute Cary, NC). The model statement will include chitosan treatment and biopesticide as the independent variables and block as the random variable. Statistical significance will be assessed at p < 0.05 and a Tukey HSD Post-hoc test will be used to separate the means.

Field evaluation of disease suppression. Chitosan treatments and microbial biopesticides will be evaluated in two on-farm orchard trials. These on-farm trials will be established through the assistance of our UNH Extension collaborators, George Hamilton and Jeremy DeLisle, who works closely with NH apple growers. Weather stations connected to the Network for Environmental and Weather Applications (NEWA) will be used to collect climate data (temperature, precipitation, leaf wetness, degree days, etc.) during these trials.

These field trials will consist of five treatments (chitosan + biopesticide 1, chitosan + biopesticide 2, biopesticide 1, biopesticide 2, and the grower’s standard as the control) applied to two apple cultivars. Each treatment will be applied to five tagged branches on each of four replicate trees (20 trees per cultivar). The chitosan/biopesticide treatments will be overlaid on the farmer’s existing spray program. If the farmers have more trees available for use in our trial, we will add additional replicates. Each chitosan and biopesticide treatment will be based on our evaluations from the controlled environment experiments. Treatments will be applied at bloom (early May) and again in mid-June. The treatments will be sprayed onto the individual branches using a backpack sprayer. The trials will rely on natural apple scab inoculum present in the grower’s orchard.

Trees will be assessed at three time points during the season (June, July, and September). Suppression of disease will be assessed by evaluation of disease severity as described by Poleatewich et al. (2012). Six leaves per replicate tree will be collected at each sampling time to measure percent leaf area affected by apple scab using WinRHIZOTM as described above.

All fruit will be harvested from each tagged branch at time of fruit ripening (around September and October). Fruit weight and quality (i.e. the presence of blemishes and sugar content) will be measured for each treatment. Fruit will be evaluated for fruit scab severity using a 0-to-6 rating scale as described by Poleatewich et al. (2012). Then, ten fruit per treatment will be randomly sampled to be used to measure Brix (total soluble solids) for sugar content using the Hanna digital refractometer (model H196801) (Hanna® Instruments, Woonsocket, RI). Each fruit will be cut into pieces for replication (20 replicates/treatment). Fruit pieces will be pressed onto the digital refractometer and the refractometer will be cleaned after every sample.

Disease severity on leaves and fruit, fruit weight, and fruit quality for each farm will be analyzed for statistical significance using a Three-Way Analysis ANOVA in JMP Pro 14 (SAS Institute Cary, NC). The model statement will include chitosan treatment, microbial biopesticide, and cultivar as the independent variables and tree (a biological replication that represents a block) as the random variable. Statistical significance will be assessed at p < 0.05 and a Tukey HSD Post-hoc test will be used to separate the means.

Objective 3: Investigate the potential for chitosan formulations to reduce viability of overwintering spores of Venturia inaequalis in leaf litter.

The apple scab pathogen, V. inaequalis survives from year-to-year on infected leaves that have fallen to the ground as leaf litter. New spores (ascospores) are released in the spring during periods of rain and high humidity. An important component of apple scab management is to reduce the amount of primary inoculum (ascospores) in an orchard (MacHardy, 1996). In this study we will investigate the effect of chitosan applications to the leaf litter on potential ascospore dose (PAD) and spore viability. These treatments will be compared to an application of urea, which is a common strategy to decrease primary inoculum of apple scab. 

Due to state-mandated COVID-19 shutdowns, the laboratory and field trials were unable to be completed in at the University of New Hampshire in 2020. However, our collaborator at Penn State, Kari Peter, is running preliminary trials in their research orchard following the our methods. These preliminary trials will give us data that we can then utilize for our trials in 2021 and 2022.  

Laboratory study to determine the potential of chitosan to reduce spore production. Leaf disks of 2.7 cm in diameter will be cut from apple leaves collected from our on-farm trials at the end of the season. V. inaequalis spores will be prepared as discussed in objective 1. Leaf disks will be inoculated and treated with three chitosan treatments and a water control as described by Carisse et al. (2000). Each treatment will be applied to 40 leaf disks (four leaf disks per jar, 10 jars per treatment). Ascospore counts will be determined as describe by Carisse et al. (2000) and counted using a hemocytometer. This trial will be repeated twice. Ascospore production will be analyzed for statistical significance using a One-Way ANOVA in JMP Pro 14 (SAS Institute Cary, NC). The model statement will include treatment as the independent variables and the jar as the experimental unit. Statistical significance will be assessed at p < 0.05 and a Tukey HSD Post-hoc test will be used to separate the means.

Field study to determine the potential of chitosan to reduce spore production under natural conditions. This field study will be conducted using the methods outlined by Carisse and colleagues (Carisse et al., 2000) with a few modifications. Apple leaves infected with V. inaequalis will be collected from on-farm trials at the end of the season. To standardize inoculum, leaves with an infection rating between 3-4 (as described previously) will be used in this experiment. This experiment will consist of five treatments (three chitosan, 5% urea solution, and a water control) and nine sampling units (bags of 50 leaves). After treatment application, leaves will be overwintered (during the winter of 2020/2021) in nylon mesh bags fixed to the orchard floor of both on-farm trial orchards (2 replications) in a completely randomized design. This trial will be repeated over the winter of 2021/2022, post the timeline of this grant. Ascospore production will be analyzed as described in the section above for artificial inoculation of the leaf disks.

Carisse, O., Philion, V., Rolland, D., Bernier, J., 2000. Effect of Fall Application of Fungal Antagonists on Spring Ascospore Production of the Apple Scab Pathogen, Venturia inaequalis. Phytopathology 90, 31–37. https://doi.org/10.1094/PHYTO.2000.90.1.31

MacHardy, W.E., 1996. Apple Scab: Biology, Epidemilogy and Management. American Phytopathological Society Press, St. Paul, MN.

Poleatewich, A.M., Ngugi, H.K., Backman, P.A., 2012. Assessment of Application Timing of Bacillus spp. to Suppress Pre- and Postharvest Diseases of Apple. Plant Dis. 96, 211–220. https://doi.org/10.1094/PDIS-05-11-0383

Research results and discussion:

In the preliminary growth room experiments, the apple seedlings did not show phytotoxicity after being treated with chitosan treatments up to a concentration of 1% (w/v). 

Participation Summary
2 Farmers participating in research

Education & Outreach Activities and Participation Summary

1 Published press articles, newsletters

Participation Summary

Education/outreach description:

This research was featured in a New Hampshire Agricultural Experiment Station article found here: https://www.colsa.unh.edu/nhaes/article/2020/08/applescab. Further, I have been interviewed about this work by the Good Fruit Grower magazine.

In the summer of 2021, I will survey growers at one of the twilight meetings that are organized by the NH Fruits Growers Association and UNH Extension. I will survey farmers with the following questions: 1) How big of a problem is apple scab on your orchard? 2) What did you learn today? 3) Do you currently apply biopesticides in your orchard? 4) What is the likelihood of you implementing new tools as a part of your IPM strategy?

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.