Progress report for GNE19-199
In this project, I propose to develop transfer methods of tomato foliar microbial communities effective at suppressing either bacterial spot or speck. I hypothesize that a community of microorganisms will provide increased plant health and prevent disease development to a greater extent than a single organism application, and that these microbes can be efficiently and economically transferred with dry plant material.
The specific objectives of this proposal are:
1. Develop a method for transferring communities during germination in the greenhouse. Expected outcome: This objective will identify the efficacy of foliar microbial community transfer using plant material.
2. Determine the amount of plant material required for detectable disease suppression. Expected outcome: This objective will establish the amount of plant material needed for disease suppression to inform the economic feasibility of each transfer method.
3. Assess the efficacy of plant material that is stored over an extended period of time for disease reduction. Expected outcome: This objective will indicate whether plant material can be stored long-term prior to application and still retain effectiveness.
Overall, this project will provide a method for designing a more sustainable crop system for protecting crops from diseases in the short-term, and potentially long-term in the field. This research will provide the critical data that will aid in selecting methods that could be incorporated into current growing practices in an economically feasible manner.
Tomato is an economically important horticultural crop in the Northeast (MD, PA, NJ, NY), particularly for small-scale growers, with approximately 8000 acres harvested in 2017 worth $59 million (USDA-AMS 2017). Yet, bacterial diseases of tomato, such as bacterial speck (Pseudomonas syringae pv. tomato) and spot (Xanthomonas spp.), are becoming more prominent in the Northeast region, and in particular Pennsylvania, due to shorter crop rotations and increased rainfall (Bogash 2016). Given the nature of bacterial plant diseases and the economic necessity of growing highly susceptible cultivars, there is significant reliance on chemical (e.g. fixed copper) and antibiotic (e.g. streptomycin) control measures (Vidaver 2002). However, such control management strategies have become less effective as resistance has emerged and spread through pathogen populations (Stall and Thayer 1962; Stall, Loschke, and Jones 1986; Obradovic et al. 2004). Hence, alternative approaches, such as utilizing biological control agents (BCAs), have the potential to substitute or combine with currently employed strategies to provide more robust disease control in both conventional and organic tomato production.
Field microbial collections: Asymptomatic tomato leaves were collected from Russell E. Larson Agricultural Research Center at Rock Springs, Pennsylvania in late August 2020. Three leaves from 5 individual plants (1 young, 1 mid, 1 old) were selected across the tomato growing field. All plant material was stored at 4C for 5 days.
Greenhouse tomato growth: Mountain Fresh Plus (Johnny’s Selected Seeds, ME) tomato seeds were surface disinfested (1min in 70% EtOH; 20min in a 0.1% Tween, 5.25% bleach solution; rinse 3x with autoclaved Milli Q water) and sown in 2” square 50 pod flats pots with non-sterile Sunshine Mix 4 soil. Seedlings were grown in greenhouse (Pennsylvania State University) and transplanted to 6” pots 12 days after sowing with non-sterile Sunshine Mix 4 soil. Plants were watered at soil level daily.
Leaf community inoculum: For spray inoculation of the microbial metacommunity, 25 leaflets from the collected material were placed in a 50 mL conical and completely submerged with 10 mM MgCl2 (~35-40 mL), vortex for 20 seconds, sonicated in an ultrasonic bath (Branson Ultrasonics Corporation, CT) for 15 minutes, and vortexed for 20 seconds. Leaflets were removed and placed in another conical, submerged with 10 mM MgCl2, vortex for 20 seconds, sonicated in an ultrasonic bath for 15 minutes, and vortexed for 20 seconds. Leaflets were removed. The remaining supernatant in both 50 mL conicals were centrifuged for 2 minutes at 7197 x g to pellet cells. The supernatant was extracted leaving 1 mL and up to 25 mL was placed in a new separate 50 mL conical. The pelleted cells were vortexed and combined into a single conical (total of ~2 mL). For storage at -80C, 500 ul of cells were added to a 1.8 mL cryovial (Genesee Scientific, CA) with 500ul of 80% glycerol and 500 ul were filtered through a 0.22 uM polyethersulfone membrane. The extracted supernatant (~50 mL) was added to the remaining ~1 mL of cells for spray application. A misting spray bottle was filled to a 1:2 ratio with 10 mM MgCl2 (cells:MgCl2). Based on total volume 0.001% Silwet L-77 was added and misting spray bottle was shaken to mix thoroughly.
Pathogen inoculum: Pseudomonas syringae pv. tomato DC3000 (Pto) was grown in liquid King’s B (KB) media at 28C for 12 hours. The cells were then centrifuged at 7197 x g, washed with 3 mL of 10 mM MgCl2 twice, and resuspended in 1 mL of 10 mM MgCl2. Bacterial suspensions were standardized to an optical density at 600 nm of 0.3 (10^8 CFU) in 300 mL of 10 mM MgCl2. Add cells to misting spray bottle with 0.001% of Silwet L-77 and shaken thoroughly to mix.
Initial leaf community transfer: At 1.5 weeks old, 4 sets of 36 plants were sprayed with either leaf community inoculum or 10 mM MgCl2 buffer with 0.001% Silwet L-77 in a misting spray bottle (Figure 2). Plants were sprayed on the abaxial and adaxial sides of the leaves until run-off at 9pm. All plants were immediately placed randomly in a high-humidity chamber for 15 hours (90-100% RH). After 15 hours, the plants were moved to a greenhouse bench under growth lights (400-watt High Pressure Sodium Crop lights, P.L. Light, Canada) on a 16 hr light and 8 hr dark cycle and misted with tap water.
Pathogen inoculation: Three days following leaf community transfer, one half of plants with leaf community and those with 10 mM MgCl2 were spray inoculated with Pto pathogen on the abaxial and adaxial sides of the leaves until run-off at 9pm at night (Figure 2). All plants were placed in the high-humidity chamber for 15 hours, then moved back to the bench and misted with tap water.
Disease assessment: Disease severity was recorded on quasi-continual scale (0-100%) for all replicate plants based on visually observed symptoms on days 0, 2, 4 and 7 post-transfer (Figure 2). Disease incidence was monitored on the presence or absence of disease symptoms at the end of each passage.
Passaging lines: A set of 36 plants represented a single passaging line indicated as C107, C108, C109 and C110. Within each passaging line, 9 replicate plants were separated into 4 treatments based on application of leaf community inoculum or 10 mM MgCl2, and/or Pto: leaf community and Pto, leaf community only, Pto only, and buffer only (Figure 3). For each treatment within a passage line, 3 plants were placed randomly across 3 blocks on a single greenhouse bench. At the end of each passage, all treatments within each passage line were processed individually and transferred every 7 days post-transfer for 10 passages.
Figure 2. Diagram depicting the transfer of field collected microbial community in a passaging experiment and the continual passaging methodology for all passage lines. Tomato leaves from a research farm were sourced for the starting microbial community. This community was sprayed onto 1.5-week-old tomato plants grown in the greenhouse. Three days after community transfer, plants were challenged with bacterial speck pathogen. After 5 days, 3 replicate plants showing the lowest disease severity were collected as the source microbial community for the next passage.
Figure 3. Detailed outline of the multiple treatments that are independently passaged within each passage line. From the starting microbial community, multiple passage lines are formed that each include four treatments: leaf community and Pto (community transfer followed by pathogen inoculation), leaf community only (community transfer followed by buffer), pathogen only (buffer transfer followed by pathogen), and buffer only (buffer transfer followed by buffer). Each treatment is independently transferred from passage to passage within each of the passage lines.
Microbial sampling and passage transfer: Seven days post initial leaf community transfer, all leaves of 3 tomato plants from the ‘leaf community and Pto’ and ‘leaf community only’ treatments with the lowest disease severity were collected. Additionally, all leaves of 3 plants from ‘Pto only’ and ‘buffer only’ were randomly selected and collected. All plant material were stored at 4C for 9 hours. Each treatment for all passage lines were processed individually following the protocol outlined for the leaf community inoculum above. The prepared leaf community inoculum was sprayed onto 9 new 1.5-week-old tomato plants with a misting spray bottle (Figure 1). Plants were sprayed on the abaxial and adaxial sides of the leaves with a single treatment within a passaging line until run-off at 9pm. All plants were immediately placed randomly in a high-humidity chamber for 15 hours (90-100% RH). After 15 hours, the plants were moved to a greenhouse bench under growth lights on a 16 hr light and 8 hr dark cycle and misted with tap water. The passage transfer was followed by the protocols for pathogen inoculation and disease assessment as stated above.
Statistical analysis: Based on the experimental set up where each passaging line was independently passaged and repeatedly measured the disease severity at the end of each passage the data was treated similar to that of a time series experiment. The disease severity at the end of each passage for all treatments were analyzed in a mixed model for repeated measures (MMRM) and post-hoc Tukey ANOVA multiple comparison using JMP® Software (SAS Institute Inc, NC).
- Develop a method for transferring communities during germination in the greenhouse. This objective will identify the efficacy of foliar microbial community transfer.
We investigated whether it was possible to transfer, establish and maintain a field collected metacommunity (the combined community from multiple plants in a single field) on tomato plants germinated in the greenhouse during August and November 2020. The method of choice for this season of tomato growth was a spray application. Briefly, leaf material was submerged in buffer and sonicated at a high frequency to disrupt the leaf attached microbes and then using a spray bottle sprayed on naïve plants (no additional applications) in the greenhouse. At the end of each passage, the resultant plants were selected, and the microbial communities were collected using the same method. The communities were then transferred using the same spray application beginning a new passage for each passage line. This method proved easy to apply and practical for the number of replicates under these experimental settings. Confirming the efficacy of this method of foliar microbial community transfer will be investigated in another project that aims to sequence the field and passaged communities to identify the microbial diversities.
- Determine the amount of plant material required for detectable disease suppression. This objective will establish the amount of plant material needed for disease suppression to inform the economic feasibility of each transfer method.
After ten passages, the effects of a spray application in the passaging method on disease severity (the average percentage of symptom coverage across all replicate plants for each treatment) and disease incidence (the average percentage of diseased plants) were observed and analyzed. The pooling of 25 leaflets from 3 tomato plants to be the source of each microbial community transfer appears to support the passaging method to detect disease suppression across the four passage lines during August and November 2020. This is based on the observed progression of disease severity following a similar trend observed in suppressive soils where disease severity increases in a field after pathogen in present in the soil, a disease outbreak occurs, and then there is a large decline in disease severity and remains low for many seasons/years (Raaijmakers and Mazzola, 2015). Disease incidence was 100% for all passages of leaf community and Pto and pathogen only treatments in all passage lines (C107, C108, C109 and C110).
Disease severity was recorded on a continuous scale for all treatments within each passage line. Overall, the leaf community and Pto treatment reduced severity of P. syringae pv. tomato infection when disease severity data was compared with the three treatments across the series of passages in a repeated measure mixed model analysis (F = 30.138; P = 0.0001). However, the leaf community and Pto was not significantly different to pathogen only treatment at each passage in an ANOVA post-hoc Tukey test. For passage line C107, disease severity increased from passage 1 with a peak at passage 4 for both leaf community and Pto and pathogen only treatments (Figure 4A). From passage 4 onwards, disease severity declines by 30-35% by passage 10. Additionally, passages 7 to 10 have an average disease severity lower than the initial disease severity presented at passage 1 for leaf community and Pto and pathogen only treatments. We can observe a peak and decline in disease severity, and that the decline is continuous for multiple passages similar to some suppressive soil examples. Treatments that did not introduce pathogen (leaf community only and buffer only), remained at 0% disease severity across all passages indicating no contamination of pathogen in the greenhouse.
Figure 4. Disease severity for each passage line represented as percentages across all ten passages with (A) passage line C107, (B) passage line C108, (C) passage line C109, and (D) passage line C110. Disease severity was recorded on a quasi-continual scale (0-100%) based on visual observations at the end of each passage for all replicate plants in each treatment and averaged.
Passage lines C108 and C109 both follow a similar trend to C107 with disease increasing and peaking at passage 4 and continuing to decline until passage 10 (Figure 4B and 4C, respectively). In comparison with the other three treatments, the leaf community and Pto treatment over time reduced severity of P. syringae pv. tomato infection in a repeated measure mixed model analysis (F = 30.196; P = 0.0001 and F = 14.795; P = 0.0001, respectively). However, there are slight differences for both C108 and C109 passage lines when compared to C107. For C108, between passage 3 and 5 there are no differences between leaf community and Pto and pathogen only treatments. The disease severity decline is also sharper from the peak at passage 4 to passage 6 and overall declining by 28% by passage 10 for both treatments. For C109, there are significant differences between leaf community and Pto and pathogen only treatments for passages 2 to 4 (P = 0.002) indicating significant decrease in disease when a field community is applied in this experimental set up. However, after the peak in disease severity at passage 4, this difference disappears as the disease severity for both treatments that include pathogen become similar in percentage. There was no disease severity recorded for leaf community only and buffer only across all passages.
The average peak disease severity for passage line 110 is at passage 5 rather than passage 4 which was observed in the other three passage lines (Figure 4D). In addition, the peak is the lowest across all passage lines at 31% for pathogen only and 26.7% for leaf community and Pto. Statistical analysis with repeated measures showed indicated significant differences between all four treatments across the entire series of passages (F = 13.3628; P = 0.0001). After peak disease severity a decline was observed within a single passage (passage 5 to 6) for leaf community and Pto by 14% and pathogen only by 18%. From passage 6 onwards, disease severity remains lower than the initial average at passage 1. The disease severity across all passages remained at 0% for both leaf community only and buffer only.
Education & Outreach Activities and Participation Summary
Presented research at Penn State Univerisity Department of Plant Plant Pathology and Environmental Microbiology’s Fall 2020 Seminar Series.
Presented research to the R&D department at Indigo Ag (agricultural technology company) seminar series Jan 2021.
The results currently show that following serial passages of a pathogen with a field collected community were able to reduce disease severity compared to passages of pathogen only. Additionally, the experimental set up of passaging matches with what is known regarding certain suppressive soils, where disease pressure builds over successive seasons of growing the same crop, followed by a sharp decline in disease that remains low over time.
We have identified that a microbial community-based approach could improve plant productivity due to increased pathogen suppression and this approach is supported by previous theoretical and empirical research. This management strategy will support sustainable IPM systems and be applicable in conventional and organic tomato production.