Progress report for GS20-225

Project Type: Graduate Student
Funds awarded in 2020: $12,347.00
Projected End Date: 08/31/2022
Grant Recipient: University of Florida
Region: Southern
State: Florida
Graduate Student:
Major Professor:
Lorenzo Rossi, Ph.D.
University of Florida
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Project Information


Huanglongbing (HLB; also known as citrus greening) is a bacterial disorder that is severely reducing global citrus production. It is caused by the bacterium Candidatus Liberibacter asiaticus (CLas) and vectored by the Diaphorina citri psyllid. It was first detected in Florida in 2005 and is now widely distributed throughout the commercial citrus-growing regions. As of today, there is no cure for HLB, and there are no commercial citrus cultivars, varieties, or scion-rootstock grafting combinations with natural resistance to infection. Thus, searching for alternative mitigation strategies is an urgent priority for a sustainable citrus industry. Anecdotal reports from Florida growers have claimed that citrus growing within the drip line of large oak trees have minimal HLB symptoms, while citrus trees nearby but not under the oak tree drip line have severe symptoms. As a result, some growers are now using oak mulch in their citrus groves, and they noticed an overall increase in production. Following these observations, the main objectives of this study are (i) to study the capability of oak mulch to contain and suppress CLas, (ii) to measure the effect of oak mulch on HLB-affected citrus physiology, and root growth and development, and (iii) to study the effect of oak mulch on the rhizobiome. Soil, leaf and root samples will be collected monthly, along with root images. The products of this research will lead to the reduction of traditional treatment methods, while improving citrus industry sustainability and productivity and ensuring continued availability to the public of healthful citrus products.

Project Objectives:
  • Study the capability of oak mulch to contain and suppress CLas
  • Measure the effect of oak mulch on HLB-affected citrus physiology and root growth and development
  • Study the effect of oak mulch on microbial life biodiversity within the rhizosphere (rhizobiome)



Materials and methods:

This project aims to apply oak bioactive materials (mulch) in a CLas-infected citrus grove. We have reached an agreement with a cooperator offering the use of a part of their existing citrus grove for this project. The study site is part of a larger orange grove, and the trial will utilize only Valencia oranges on US-812 (4 years old). This rootstock/scion combination was selected for its sensitivity to low soil and water quality and because is the typical combination used by growers across the Indian River Region. The experimental design for the study will utilize a randomized complete block design where the main variable will be the presence of oak mulch (300 kg of muck per plot). All other grove management operations, including fertilization, irrigation, and pest control will occur under standing operating conditions determined by the grower cooperator. Each plot will have 15 trees to be used for canopy growth assessment, root/tree health, disease severity/incidence, soil and leaf nutrient characterization, and soil microbial activity assessment. All treatments will be replicated 6 times.

Oak mulch will be obtained from a landscape company and it will be chipped and applied to the field at the beginning of the experiment. A second mulch application will be conducted 1 year after the start of the project. Lastly, measurements for this project include a wide range of soil physical, chemical, and biological parameters, root parameters, tree physiological assessments, and rhizosphere observations. The measurements and methods pertaining to each objective are discussed below:

Progress: Mulch was applied in to field plots in September 2020 as scheduled. 

Objective 1Study the capability of oak mulch to contain and suppress CLas in vivo under field conditions.

Leaf CLas titer will be taken at the start of the project and continue monthly for the duration of the project. Leaf will be collected, and the microbial DNA will be extracted using the Qiagen DNeasy kit. Changes in individual tree CLas quantities will be tracked and analyzed throughout the duration of the experiment. Tree data will be analyzed using SAS to determine if there are treatment effects among the two treatments.

Progress: Leaf titer has been collected monthly since mulch application. Root titer analysis has been added to this objective and will be collected every 3 months.

Objective 2Measure the effect of oak mulch on HLB-affected citrus leaf physiology and root growth, development and physiology.

Root growth will be assessed using mini-rhizotrons installed in each plot (at least 2 per plot) at a 45° angle throughout the root system. A CID-root imager camera (CID Bio-Science, USA) will be used to take root scans and estimate root diameter, length and overall root growth. Root health will be assessed using minirhizotron scans and root collections in combination with WHINRhizoTRON analysis software (Regent Instruments Inc., Canada). In addition, leaf and soil nutrient concentrations (N, P, K, Ca, Mg, S, B, Zn, Mn, Fe, Cu)  will be determined at the start of the project and at approximately 1-month intervals for the duration of the project. Samples will be collected and sent to Waters Ag Laboratories in Camilla, GA for analysis of nutrients, pH and CEC. Finally, soil moisture analysis between treatments will be taken monthly.

Progress: Mini-rhizotrons were installed in September 2020 and initial root scans were taken. Scans continued until December 2020. Scans for January- March  2021 were not conducted due to mechanical and calibration issues with the scanner. Scans are expected to resume in April 2021. Leaf and soil nutrient data has been collected monthly. Root nutrient analysis has been added to this objective, and roots have been sampled with soil and leaves monthly. Additionally, root density measurements have been added to this objective. Root density will be measured every 3 months. 

Objective 3Study the effect of oak mulch on microbial life within the rhizosphere (rhizobiome).

 Root and soil microbial activity (respiration) will be characterized using a LI-COR 6800 (LI-COR, USA) connected to a soil CO2 analyzer. Respiration will be measured every 3 months for the duration of the project. Additionally, a subsample of soil and mulch will be collected for rhizobiome analysis. Soil samples containing citrus roots will be collected at the beginning of the experiment, after one year and at the end of the field trials. The microbial rRNA will be extracted from soil samples using the Qiagen DNeasy PowerLyzer PowerSoil Kit and soil microbial diversity and composition will be assessed using high-throughput amplicon sequencing. Extracted rRNA will be sent for high-throughput amplicon and shotgun metagenomics sequencing to the University of Illinois Chicago DNA Services Center. Data will be analyzed in QIIME 2, Phyloseq and Spieceasy R packages, and PICRUSt. Machine learning algorithms will be used to identify specific taxa responsible for differences among treatments and determine their importance in correlation with the measured soil nutrient and tree health parameters.

 Progress: Soil and mulch were collected for rhizobiome analysis. Root and soil respiration has not begun due to  complications obtaining the Li- Cor as a  result of USDA coronavirus shutdowns. Soil respiration measurements are expected to begin in summer 2021. 

Research results and discussion:

Preliminary results by objective

Objective 1: No differences in leaf titer have been observed between treatments. All trees in the study have HLB and are symptomatic. 

Objective 2: Oak mulched plots have shown increased root growth in both mini-rhizotron images and root density. Soil analysis shows increased phosphorus, potassium, and magnesium in mulched plots compared to non mulched plots. No differences have been observed in leaf nutrient contents, soil pH, and soil cation exchange capacity between treatments. 

Objective 3: Data from microbiome analysis has not yet been analyzed, however, more biological activity (soil fauna and flora: mushrooms, worms, etc) has been visually observed in mulched plots. 

Participation Summary

Educational & Outreach Activities

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

Participation Summary

10 Farmers
10 Ag professionals participated
Education/outreach description:

Gave a 30 minute presentation to between 20-40 growers, extension, and industry professionals at the Central Florida Citrus Under Protective Screening (CUPS) Field Day. This event was organized by the Volusia County extension and took place at Vo-LaSalle Farms, Inc (De Leon Springs, Fl)  on March 25, 2021. Topic included rationale behind the oak mulch study, experimental design/ materials and methods, and preliminary results of the study. Other outreach includes published press articles in Citrus Industry Magazine, News4Jax, MorningAgClips, and UF/IFAS blogs. 

Project Outcomes

Project outcomes:

Ideally, this project will contribute to the knowledge regarding the use of mulch in the citrus industry. Economically, if the addition of mulch increases plant available nutrients, growers may be able to use less synthetic fertilizers, contributing to economic and environmental sustainability. 

Knowledge Gained:

The project began in September 2020 and since, our knowledge, skills, and awareness of sustainable agriculture has increased. In only 6 months, improvements in soil nutrient levels due to the application of mulch has been evident. We originally hypothesized that changes in nutrient levels may take years to manifest, however, these preliminary results have taught us that the breakdown of mulch happens much quicker than anticipated. 

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.