Investigating Lobster Byproducts as Soil Amendments for Disease Suppression and Soil Health Improvement in Potato Production

Progress report for GNE22-277

Project Type: Graduate Student
Funds awarded in 2022: $14,620.00
Projected End Date: 08/31/2024
Grant Recipient: University of Maine
Region: Northeast
State: Maine
Graduate Student:
Faculty Advisor:
Dr. Jianjun Hao
University of Maine
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Project Information

Summary:

Maine is a unique state with a wealth of agricultural production amidst the lobster industry. While these two economies are distinct, the use of lobster processing byproducts as a soil amendment could benefit both industries. Potato tubers are constantly interfacing with both soilborne pathogens and beneficial microorganisms and are subject to over 85 different diseases. Post-processed lobster shells can be finely ground to produce lobster shell meal (LSM). This product contains chitin, much like the key components of fungal cells and their overwintering structures. Adding chitin-rich material such as LSM can increase the populations of some chitin-degrading microorganisms that also suppress soilborne pathogens. We propose to use LSM as a soil amendment to enhance soil health by promoting beneficial microorganisms for disease suppression. In a greenhouse, the efficacy of LSM will be determined using potato ‘Shepody’ and inoculated with either no pathogens, Verticillium dahliae, Rhizoctonia solani, or Streptomyces scabies, three soilborne pathogens. In the following year, a field trial will be performed to determine the impact of LSM on potato production, utilizing soil inoculated with V. dahliae, and chemical fumigation. Potato yield, disease incidence and severity, and shifts of microbial communities between treatments will be examined. This study aims to improve the understanding of how plant diseases may be suppressed by adding LSM to soil for modifying microbial communities. Additionally, the use of LSM presents an opportunity to connect the potato and lobster industries in Maine and transform shellfish industry byproducts into a highly valuable resource.

Project Objectives:
  1. Determine the efficacy of LSM for promoting beneficial soil microbial communities and reducing soilborne diseases of potato
  2. Analyze soil microbial community dynamics under LSM treatment
Introduction:

     The purpose of this project is to reduce disease-related losses in potatoes by enhancing soil health with the addition of a shellfish industry byproduct. Potatoes are the most valuable crop in Maine and have been part of the economy since the mid-1700’s (USDA 2021; Johnston 1972). In 2019, 51,500 acres of potatoes totaling a yield of 16,738,000 hundredweight were grown in Maine (USDA 2020). Unfortunately, the potato industry has been challenged by the ever-present issue of soilborne diseases. 

     The first problem to be addressed is decreasing the impact of soilborne diseases and consequently increasing yields of potatoes. Soilborne diseases have been managed in various strategies, including using tolerant or resistant varieties, chemical treatments, and soil fumigation. They also can be managed with the addition of soil amendments which can stimulate disease-suppressive beneficial microbial communities in addition to improving soil quality and nutrients (Larkin 2015). 

     Depending on the source, soil amendments have different functions and effects on soilborne pathogens and potato (Hao and Ashley 2021). Therefore, it is critical to understand how the amendment works before using it. An amendment such as LSM contains a high concentration of chitin, which provides nutrients for microbes which degrade and utilize chitin for their growth (Andreo-Jimenez et al. 2021). Some of these microorganisms can suppress plant pathogens with one or more modes of actions, such as directly degrading pathogen cell walls or overwintering structures that contain chitin as a major structural component (Ramirez et al. 2010). This increase to beneficial microbial communities through the introduction of organic matter could also be described as an improvement to soil health. Soil health is a critical factor of sustainability and longevity of agricultural lands. In order for farmers to continue practicing agriculture for generations to come, sustainable practices which contribute to soil health and subsequent plant health are of utmost importance (Hao and Ashley 2021; Larkin 2015). Agricultural practices which rely on the existing ecology of microbes in the soil to assist in disease suppression as opposed to solely chemical application methods encourage this longevity and reduce the impact on the environment caused by chemical application. 

     The second problem which is being addressed in this study is with the innovative utilization of lobster byproducts post-processing to remove them from the waste stream. Disposing of lobster shell waste is highly costly, for example, a facility processing 15,000 pounds of lobster per day can expect to pay upwards of $4000 per month for disposal services. A small portion of lobster shell waste is composted, but the majority is sent to landfills (Fulton et al. 2013). Maine is uniquely positioned as a state with a wealth of agricultural production amidst the highly valuable lobster industry. The use of lobster processing waste as a soil amendment could help build collaboration between these two vital industries in the state of Maine. Overall, this study will address two problems with a solution which advances sustainable agriculture and longevity of agriculture through soil health.

Cooperators

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  • Curt Brown (Educator)
  • Robert Bayer (Educator and Researcher)
  • Luke Holden (Educator)

Research

Materials and methods:

The lack of field research and technical application of shellfish byproducts in agriculture indicates that field research is of most use. However, because of the intrinsic challenges and large scale of field research, preliminary studies took place in the greenhouse located at The University of Maine in Orono. Following the greenhouse trial, a field trial took place. Lessons learned in the greenhouse trial aided in determining the best direction for field application, experimental design, and rates.

  1. Effects of lobster shell meal (LSM) on soilborne pathogens and potato plants, Preliminary greenhouse studies. In the University of Maine greenhouse, the potato variety ‘Shepody’ was planted in a mix of garden soil and potting soil (1:1) in one-gallon pots in March and July of 2022. ‘Shepody’ was selected because it is susceptible to both V. dahliae and S. scabies. The soil was inoculated with either Verticillium dahliae or Streptomyces scabies inoculums, the causal agents of potato early dying (PED) and common scab, respectively, and non-inoculated soil was used for a non-diseased control. Prior to planting, inoculum for S. scabies was made by growing the bacteria in vermiculite mixed with liquid medium (Say’s solution: 40 g sucrose, 2.4 g asparagine, 1.2 g K2HPO4, 20 g yeast, 1 L sterile water). The bacteria and vermiculite combination was added to the soil in pots at a rate of 1:10 inoculum to soil, and mixed thoroughly. The final inoculum concentration was 105 colony forming units (CFU) per gram of soil. For V. dahliae inoculum preparation, microsclerotia, the rigid overwintering structure of V. dahliae, were used. Twenty microsclerotia per gram of soil were added to pots by suspending microsclerotia in sterile water and adding to vermiculite. Lobster shells collected from local processors were dried and ground using a grain mill to produce a fine powder, which was used as the standard LSM. Soil samples were collected and stored in a -80°C freezer until further processing to represent soil before adding potato seed pieces, pathogen inoculum, or LSM. LSM was then blended into the soil at various rates (0%, 2%, and 8% w/w). These ranges were selected based on previous studies and to help narrow in on appropriate rates for subsequent work. There were initially five pots per treatment within each pathogen inoculation category (V. dahliae, S. scabies, or uninoculated) and both planting groups. In the first planting group, the pots were immediately planted with sprouting seed tubers, and the second group remained without tubers for the first three months before planting with sprouting seed tubers. This gave an indication of how microbial communities are changing without the addition of plant tissues, but with the addition of LSM alone, and model a fall or early spring amendment timeline. This also gave time for microbial communities to respond to the addition of LSM. Pots were set up and randomly arranged in the greenhouse bench. Plants were watered as needed throughout their growth and until the plants began to naturally senesce around 3 months after planting.

            Plant heights were measured over the growing season until the natural death of the plants occurred, after which, plants were harvested. To harvest the plants, plants were cut at the soil line and all plant tissue were placed into paper bags to dry in a heated room for approximately one week, or until the plant matter was fully desiccated. Desiccated plant matter was weighed and recorded as aboveground biomass. A few days after the removal of aboveground plant tissue, tubers were removed from the soil, the surfaces were cleaned of any residual soil, and they were processed immediately. The total weight of the tubers, tuber numbers, and disease symptoms were recorded. Disease incidence described the presence or absence of symptomology and disease severity was recorded in various ways depending on the disease. To determine PED infection symptom severity, tubers were cut in half longitudinally, the percent of the vascular ring which was discolored was recorded. To determine the severity of symptoms of infection by S. scabies or Rhizoctonia solani, the causal agent black scurf, the percent of the tuber surface which was covered by scabs or sclerotia, respectively, was recorded. Indigenous populations of R. solani produced disease symptoms in tuber without inoculating the soils, and this was recorded as well.

            A follow-up greenhouse study was performed in March and July of 2023. Six treatments with five replications were established, building upon challenges associated with the first greenhouse trial. Fertilizer was added as a treatment factor and inoculum was combined in equal parts from R. solani and V. dahliae to ensure proper inoculum levels to cause disease, and is described below. Treatments were once again prepared twice to simulate a spring and fall amendment timeline as described in greenhouse trial 1. To best compare to the field trial, rates of inoculum, fertilizer, and LSM were all as similar as possible to those used in the field trial. The rate of inoculum was 20ml/foot, and as the pots were 8” in diameter, a rate of 14ml/pot was used. Virgin grain was added at the same rate to non-inoculated pots. The rate of fertilizer (14-14-14) was applied at a rate of 1100lbs/acre, typically incorporated at a depth of 12”, so each pot received 2.99grams of fertilizer. The rate of LSM was described by Coast of Maine to be applied at a rate of 5lbs/100ft2. In the field, the LSM was incorporated to a depth of approximately 4”, resulting in a rate of approximately 1.7% w/w. Data was collected in the same fashion as in the first greenhouse study, with the exception of plant height, which was not collected.

Greenhouse preliminary trial
Plants growing in the initial greenhouse arranged by disease inoculum and LSM rate.

 

     Field Studies. Following the greenhouse studies, two field trials took place in Presque Isle at the Aroostook Research Farm, Maine. Potato ‘Russet Burbank’ was planted in three rows within 25-foot-long plots with four replicates. Soil treatments included unamended soil, unamended soil inoculated with V. dahliae, LSM amendment alone, LSM amendment with V. dahliae inoculum added, soil fumigation with metam sodium at 50 gal/A amended with LSM and inoculated with V. dahliae, and soil fumigation with metam sodium at 50 gal/A inoculated with V. dahliae. These additional treatments gave a comparison to typical potato agricultural practices. The fumigation was performed in the fall of 2022 under favorable weather conditions. These trials took place in adjacent fields which have not had exposure to LSM previously and had been planted in oats for at least one year prior to these trials. Lobster shell amendments were applied in the fall of 2022 before planting seed tubers in the spring. The rate of LSM as an amendment was informed by industry standards provided by Coast of Maine, a compost and soil amendment company. The rate of LSM for the field trial was consequently 5lbs per 100 ft2 (approximately 0.35-0.7% of the soil % w/w). LSM was also procured from Coastal Chitin, LLC, a local processor, which aided in the industry collaborations and simplified the process of preparing large volumes of LSM. This prepared LSM was received pre-dried, pre-ground, and ready to use. The field was maintained during the growing season using standard operational practices, including fertilizer application and pest control. The fields were irrigated with rainwater only. This field evaluation also served as a demonstration plot, because of its location at the research farm, and was shared with farmers at the annual summer meeting. A second field trial was established in the spring of 2023 to simulate a spring amendment timeline. However, because of the difficulty of fumigation in the spring, this factor was not included in this trial. Consequently, the experimental design was simpler and consisted of four treatments: unamended and not inoculated, unamended and inoculated, amended with LSM only, and amended with LSM and inoculated. Soil was sampled at planting, mid-season, and before harvest.

     At harvest, potato yield, disease incidence and severity of PED were evaluated to determine the impacts of these treatments. Additionally, incidences of hollow heart, a physiological condition, were noted if tubers had hollowing in their core. Data was analyzed using similar methods for both greenhouse field studies. Statistical analyses were performed using the R package Agricolae. Effects of treatments on disease incidence, disease severity, and plant biomass were determined using analysis of variance and Fisher’s Least Significant Differences tests.

LSM before being incorporated into the soil in the fall of 2022.
LSM before being incorporated into the soil in the fall of 2022.
  1. Soil microbial community dynamics analysis. In the trials described in objective 1, field soil samples were collected in the fall-amended trial after fumigation, then in both before planting, in the middle of the growing season, and before harvest. Each soil sample consisted of a minimum of ten cores randomly selected in the plot which are homogenized in a gallon size Ziploc bag to give a representative composite sample of the plot. This helped us catalog changes to the microbial community over time. Soil samples were sieved and stored at -80°C until processed. DNA was extracted using 0.25 grams of soil and a DNEasy PowerSoil Pro Kit according to the provided instruction. DNA was quantified, and purity will be ensured before sending for sequencing analysis at The University of Michigan Genomic Sequencing Facility. Samples were processed using NextGen sequencing on an Illumina MiSeq to return sequencing reads which were used to identify fungal and bacterial community members. Amplicons of the samples were generated using the V3-V4 hypervariable regions of 16S rRNA region to determine the presence of various bacteria present in the soil samples. For fungal populations, the ITS2 rDNA region was amplified (van Horn et al. 2021). 

     Raw data of Illumina sequencing was processed to filter sequences, remove chimeras and other unwanted contaminating sequences, and clustered into amplicon sequence variants (ASVs) at 97% sequence similarity, which was analyzed using the DADA2 pipeline. R packages such as Phyloseq, DeSeq2, and Microbiome were used to quantify and visualize community differences between samples and treatments. Microbial diversity was measured by a series of ASV-based analyses of alpha and beta diversity (Rosenzweig et al. 2012; Rajendhran et al. 2011). Community differences were visualized using ordinations of Bray-Curtis and Jaccard dissimilarity (van Horn et al. 2021; Callahan et al. 2016). Core members which were unique to treatments were also called and described, should they match a previously described organism to genus and species (Callahan et al. 2016). Differential abundance of microbes will be performed using DeSeq2 to determine if specific ASVs or phyla are enhanced or reduced following our treatments (Love et. al 2016). 

Research results and discussion:

In the initial greenhouse trial, the aboveground biomass of potato plants increased significantly with increasing rates of LSM, regardless of if plants were inoculated with a fungal or bacterial pathogen, or not inoculated at all (p=2e-16; figure 1). However, the highest aboveground biomass was seen in non-inoculated plants with the highest rate of LSM. The number of tubers was not significantly different between treatments, but this could also be an artifact of the small volume of soil in which the plants were being grown (p=0.26; data not shown). Plants that were not inoculated grew taller overtime when amended with the highest rate of LSM, as is seen in figure 2 (p=4.11e-7). Disease severity was inconclusive from this initial greenhouse trial and no significant differences were seen in pots inoculated with V. dahliae or S. scabies (p=0.152 and 0.251, respectively); however, one trend was noticed. While not significant, disease severity overall was lowest in the treatments which were amended with the lower rate of LSM compared to non-amended or the higher rate of LSM. Severity of PED, as measured by the percent of the tuber vascular ring which was discolored, was also not significantly different (p=0.967). Scab severity was not significantly different, but the lowest rate of LSM had the lowest severity numerically, regardless of treatment (p=0.669; figure 3).

Figure 1. Biomass was significantly increased by the highest rate of LSM in the preliminary greenhouse trial. The number of tubers was not significantly different between treatments.
Figure 1. Biomass was significantly increased by the highest rate of LSM in the preliminary greenhouse trial. The number of tubers was not significantly different between treatments.
Figure 2. Plant growth overtime without any pathogen inoculum added. Growth overtime was highest with higher rates of LSM, and lowest in non-amended treatments.
Figure 2. Plant growth overtime without any pathogen inoculum added. Growth overtime was highest with higher rates of LSM, and lowest in non-amended treatments.
Figure 3. Disease symptoms observed following the first greenhouse trial.
Figure 3. Disease symptoms observed following the first greenhouse trial.

In the second greenhouse trial, aboveground biomass was significantly different between treatments (p=2.05e-5), and the heaviest group was the combined LSM and fertilizer treatment (figure 4). Total tuber weight and number were once again not significant, likely as a result of the highly restricted environment in which these plants were grown (p=0.693 and 0.348, respectively). Disease incidence was variable within treatments, but consistently significantly different. The incidence of PED was shown to be significantly higher than any other treatment in pots inoculated with disease and amended with both LSM and fertilizer (p=00825; figure 5). Incidence of black scurf was highest in treatments which were amended with LSM and fertilizer, however interestingly, these pots were not inoculated and indicated that this was R. solani which was naturally occurring in the soil (p=0.0027). A similar trend was seen with respect to the incidence of common scab, in that treatments which had both LSM and fertilizer, and disease and LSM had the highest incidences of disease symptoms (p=5.75e-6). It is worth noting that these two treatments were the only ones to express symptoms of common scab, and this could be related to the irregular microbial content of commercially available potting soil. Pots were prepared in batches and this could have played a role in the irregularity.

Figure 4. Aboveground biomasses of potato plants by treatment group (p=2.5e-5).
Figure 4. Aboveground biomasses of potato plants by treatment group (p=2.5e-5).
Figure 5. PED incidence in plants grown in greenhouse trial 2.
Figure 5. PED incidence in plants grown in greenhouse trial 2.

 

The fall-amended field trial, there were no significant differences in yield (p=0.059; figure 6), however there was a significant blocking effect which was driven by our replications (p=0.000109). Both PED incidence, and hollow heart incidences were not significantly different by treatment (p=0.221 and 0.999, respectively). The severity of PED was significantly different by groups, with the most severe symptoms occurring in the fumigated and inoculated treatment, and the least sever symptoms occurring in the treatments which were not inoculated and amended LSM, and the inoculated, fumigated, and amended with LSM (figure 7).

Figure 6. Total yield of fall amended treatments.
Figure 6. Total yield of fall amended treatments.
Figure 7. PED severity in fall amended treatments.
Figure 7. PED severity in fall amended treatments.

In the spring amended field trials, there were no significant differences in yield, PED incidence or severity, or incidence of hollow heart (p=0.59, 0.969, 0.894, and 0.799, respectively). While PED incidence was not significant, the highest incidence was seen in the unamended and inoculated treatment, and similarly, this treatment also had the most severe PED symptoms.

Microbial community analysis is in progress, sequences have been received and are being processed. These results will be ready in time for the final report.

Research conclusions:

There are no strong trends across this study, however there are some significant variations, which suggests that more research is needed. While PED symptoms were significantly less severe in the fall amended field trial in the treatment group which was inoculated, fumigated, and amended with LSM, this trend was not consistent across other treatments. Without further investigations, amendment with LSM may not show significant disease reductions within one growing season. The long-term effects are yet to be investigated, and with native populations of disease pressure. Finally, the impacts of LSM amendment clearly promote the development of potato plants in greenhouse settings, but this may or may not be translated to a field environment. The impact of LSM on soil microbial communities is still in progress, but this may aid in the interpretation of this novel soil amendment’s impact to soil health.

Participation Summary

Education & Outreach Activities and Participation Summary

1 On-farm demonstrations
5 Published press articles, newsletters
1 Webinars / talks / presentations

Participation Summary:

10 Farmers participated
10 Number of agricultural educator or service providers reached through education and outreach activities
Education/outreach description:

     Aside from publication which mostly extends outreach into the scientific community, presentations and factsheets will be created and distributed to farmers which may benefit from the results of this study. Presentations will take place at field days and other industry meetings as appropriate. Additionally, to fully ensure the viability of this project, contact with soil amendment companies and processors is essential to ensure that not only the results of this study, but the actual soil amendments are available to farmers, should they prove useful. We would work closely with The University of Maine Business School, two different soil amendment companies who are current collaborators, the lobster industry, and sustainability groups to develop an effective distribution pipeline of information and soil amendments.

     We have been featured in three written news articles, one newsletter geared towards the lobster industry, and one video news segment, links to which are included below. Additionally, a public seminar was given at The University of Maine in December 2022 detailing the specifics of the project.

 

Project Outcomes

1 Grant applied for that built upon this project
1 Grant received that built upon this project
$1,500.00 Dollar amount of grant received that built upon this project
1 New working collaboration
Project outcomes:

No outcomes have been achieved yet, but there certainly has been a lot of interest from the public. The estimated number reached is a low estimate and takes into account the individuals whom we communicated with and formed relationships following the news reports. This number does not include the large volume of individuals whom were reached who did not follow up concerning the project. 

Knowledge Gained:

So far in this project, we have learned the delicate nature of collaboration and have worked hard to maintain healthy working relationships. We encountered challenges with respect to producing the volume of LSM needed for the field trial and our collaborators within the lobster industry were instrumental in successful preparation for the 2023 field trial. We have also been featured in a number of news reports and have initiated contact with many other potential collaborators. This is a critical aspect of sustainable agriculture because collaboration and communication are essential for successful adoption of techniques and communication of results. 

As a team, we earned an additional $1,500 undergraduate research grant from the University of Maine to support an undergraduate student who is conducting a related project utilizing LSM and compost. 

Assessment of Project Approach and Areas of Further Study:

The amount of data which would not normally be collected in a plant pathology study contributed to insights into the growth and productivity of potato plants in the greenhouse, especially as they were likely hindered by growing in small, one-gallon pots compared to field conditions. In the future, our greenhouse design will need to be altered, but in the short term, the results provided valuable insight with respect to the experimental design of the field trial. 

Information Products

    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.