Progress report for GS22-254
Project Information
Consumer preference is growing in organic field production of vegetables, especially tomato, peppers, and cucurbits. In Florida, organic production of peppers involves crop rotation with squash and soil amendment with compost. These practices are critical for various aspects of sustainability that address soil management and soil-borne plant parasitic nematodes. Optimization of management practices can reduce limitations of crop productivity by plant parasitic root-knot nematodes (RKNs; Meloidogyne spp.). Although studies demonstrate that compost can suppress RKN, many aspects of how compost does this lack experimental verification. The use of RKN resistant vegetable varieties provides an alternative method to chemical nematicides and is suitable for organic production. Therefore, the benefits of using RKN resistant varieties include increasing yield of the respective crop and reducing RKN population densities. RKN resistant pepper varieties are available for commercial production, but there is a scarce genetic diversity regarding resistance conferred against this pathogen. The objective of this work is to test RKN resistant pepper cultivars with value for improving productivity of organic operations. We propose to test the impacts of integrating an advanced inbred variety of pepper (‘UFRJ107(6)A3’, named ‘Ruby’) into production involving crop rotation with squash in which compost application is used as a tool to both suppress RKNs and improve soil fertility. Gaining insight into the range of pepper host resistance as a supplement to cultural management practices involving organic amendments will be valuable for providing research-based recommendations to vegetable growers in the Southern United States.
1) Evaluate productivity of pepper cultivars differing in RKN resistance when integrated with soil organic amendment treatments in RKN-infested plots to measure effectiveness of genetic resistance under field conditions.
Field trials will be conducted at the University of Florida Plant Science and Research Education Unit (PSREU) located in Citra, Florida. They will be maintained in areas reserved for organic production research. Soil samples will be collected at the beginning and end of field trials to determine rate of change in RKN population density. The efficacy of organic amendments, with or without RKN resistant pepper varieties, for the control of RKN will be tested and measured through plant productivity and susceptibility to RKN. Pepper plant productivity will be measured by analysis of early and total fruit yield, fruit quality parameters, and plant nutrition during early vegetative and 50% flowering stages. Pepper plant roots will be evaluated for levels of RKN galling at the time of harvest to measure effectiveness of resistance. Organic amendment’s nutrient content, water holding capacity and culturable bacterial counts will be determined before application.
2) To test for potential nematicidal natural compounds in pepper plant roots induced by compost.
Water-soluble compounds such as phenolics have been suggested to be induced as part of systemic acquired resistance response promoted by compost (4), but the chemical properties of roots of different pepper genotypes affected by this remains unclear. Susceptible and resistant pepper plants will be evaluated for induced resistance after application of compost in a growth chamber experiment.
3) Integrate RKN-resistant pepper into crop rotations to determine effectiveness of RKN-resistance in improving yields of subsequently planted RKN susceptible vegetable crop.
Root infection by RKNs, soil RKN population, and productivity of susceptible squash planted after ‘Ruby’ will be measured. Root infection will be measured through determining galling severity for both susceptible and resistant plants at the time of harvest. Soil RKN population densities will be determined at the beginning and end of each trial with appropriate controls. Productivity of susceptible squash planted after ‘Ruby’ will be measured through evaluation of fruit yield, fruit quality parameters, and plant nutrition during early vegetative and 50% flowering stages. Rate of change in RKN soil population and galling severity will be measured for the squash plants at the time of harvest to test for suppressive effects conferred by the different sources of pepper resistance.
4) Report the results of this research to inform BMPs and plant breeders involved in production systems dealing with RKN.
The work is intended to address issues of pepper cultivar selection with relevance to economic loss due to RKN infection. Also, it will address the effect of organic amendment on both pepper plant productivity and level of RKN disease in susceptible pepper cultivars. This objective includes dissemination of information through extension outreach, conference presentations and research publications in refereed journals.
Research
Citra
The studies for objectives 1 and 3 will be conducted at the University of Florida/IFAS Plant Science and Research Education Unit (PSREU) in Citra, Florida. The site holds plot areas reserved for organic production research, which this study will utilize. This site is maintained by UF-employed personnel and provides customizable maintenance of plants .
Objective 1 - Four replicate experiments will be conducted under a completely randomized split-block design over two years. Three sub-objectives are to measure effect of genotype and compost on (a) pepper productivity, (b) RKN resistance, and (c) RKN soil population density (Table 1).
Cattle manure compost will be applied evenly on the top 20 cm of soil at a rate of 10 l m-2 for one bed measuring 1x15 m, while a second bed will serve as an unamended control. The soil nutrition contained in the unamended row will be tested, then supplemented, with N-P-K granular organic fertilizer to meet the same nutrition levels as the compost-treated row. The beds will be separated by 5 m to prevent influence of leaching into the unamended beds and covered with white plastic mulch. Chemical properties of cattle manure compost will be determined by analyzing a water extract (1:5 w/w) (16).
Four pepper varieties differing in the presence or absence of resistant genes will be examined to identify any interaction effects between compost and resistant genes on RKN reproduction. Pepper transplants will be prepared and maintained in a greenhouse until plants reach the 4-5 leaf stage (approximately 8 weeks). Seedlings will be transplanted in beds along two lines spaced 40 cm apart. Each replicate will be represented by 10 plants per genotype for a total of 3 replicates. Effect of genotype and compost treatment on productivity will be measured through various fruit yield, quality and growth parameters.
Secondly, galling index will be measured for all plants at the end (time of harvest) of each field trial. Galling index refers to a method for measuring RKN infection, which involves observing the ratio of galled root area to total root area. Gall indices of 1 to 3 will be classified as resistant, while plants rated 4 or 5 will be classified as susceptible (17).
Third, RKN soil populations will be determined at the beginning (time of planting) and end (time of harvest) of each field trial. Soil cores (1.75-cm diameter x 20-cm depth) will be collected from rootzones of six plants per block for a total of 18 samples per treatment. Second-stage juveniles (J2) will be extracted from soil (100 cm3) of each sample by the Nematode Assay Lab in the Entomology and Nematology Department at UF. RKN identity will be verified using DNA sequences amplified using specific polymerase chain reaction primers (18).
Finally, root samples will be harvested from pepper roots. Each replicate will be represented by 3 plants per genotype for a total of 3 replicates. The root samples will be homogenized and processed through a 20% methanol in water extraction. Extracts will be used for testing in objective 2.
Table 1. Parameters measured for determining effectiveness of compost and pepper genotype on crop productivity and RKN soil population suppression
|
Productivity descriptor |
Sampling Depth |
Analysis |
Frequency |
|
Fruit quality + total yield |
10 samples/block |
Total yield, fruit length, fruit width, pericarp thickness, brix, dry-to-fresh weight ratio |
Time of harvest |
|
Vegetative growth |
10 samples/block |
Plant height, plant canopy width, stem length, mature leaf length, and mature leaf width |
Time of 50% flowering |
|
RKN soil population |
1 sample/block |
RKN density (J2/100 cm3 of soil) |
Time of planting and time of harvest |
|
Resistance |
10 samples/block |
Galling index (1 to 5 rating system): 1, no infection; 2, 1-25; 3, 26-50; 4, 51-75; 5, 76-100% of roots galled |
Time of harvest |
Objective 2 –Root extracts will be prepared from plants removed at the end of the experiment conducted in objective 1. Three plants per block will be randomly selected (total 18 per variety) for root extracts. Root extracts will be prepared at the laboratory by extracting 3 g fresh weight of tissue extracted in 10 mL of 20% methanol and stored at -80 °C until use. To test for potential compounds that may inhibit nematode reproduction, RKN sensitive pepper ‘California Wonder’ seedlings grown in sand culture will be used. Each individual plant will be treated with each individual root extract preparation. Negative control plants will be treated with solvent control. One day after treating the roots with the extracts or solvent control, plants will then be inoculated with infective-stage juveniles of M. incognita. Plants will then be evaluated for susceptibility through measuring egg mass indices and reproduction factors four weeks after inoculation.
Objective 3 - Two repeated experiments will be conducted over four seasons under a randomized complete block design with a total of three replications per treatment. Each replication will consist of 10 plants per block (30 total per genotype). Plant material will be prepared and soil samples will be collected as described in objective 1.
After harvesting the pepper crop, all plant material will be carefully removed from the bed and roots evaluated for gall severity. Soil samples will also be collected to measure RKN soil population. RKN-susceptible squash (Cucurbita pepo Linnaeus) cv. Spineless Beauty seeds will be then directly sown in the same holes of the bed. When the immature fruit are ready for harvest, fruit yield, fruit quality, vegetative growth, gall severity, and soil samples will be evaluated. The data collected from the experiments described in each objective will be subjected to one-way analysis of variance (ANOVA) and mean separation tests for significance differences will be used to evaluate the treatments
Objective 4 - Recommendations will be written as fact sheets and published through the UF Institute of Food and Agriculture Science (IFAS) Electronic Data Information Source (EDIS) following peer review. Results will also be communicated at the annual meeting of Florida State Horticultural Society and Society of Nematologists during year 2. Results will also be communicated to participants in one field day held for vegetable growers.
Period covered by report: January 2023-May 2023
OBJECTIVES
The objectives of the current study are to (1) evaluate productivity of pepper cultivars differing in root knot nematode (RKN) resistance when integrated with soil organic amendments in RKN-infested plots to measure effectiveness of genetic resistance under field conditions, (2) to test for potential nematicidal natural compounds in pepper plant roots induced by compost, (3) integrate RKN-resistant pepper into crop rotations to determine effectiveness of RKN-resistance in improving yields of subsequently planted RKN susceptible vegetable crops, and (4) report results of this research to inform BMPs and plant breeders involved in production systems dealing with RKN.
INTRODUCTION
During this funding period, the focus was on objective 1 as additional objectives stem from and connect to the first. We have been preparing all the materials and procedures since Jan 2023. The series of work involved procurement of supplies such as seeds, potting media, containers, pesticides and fertilizers needed for the work, culturing and maintenance of root-knot nematode (RKN) strains on tomato plants, evaluation of pepper breeding lines and varieties for RKN resistance inside a greenhouse and in environmental growth chambers, preparation and maintenance of pepper transplants for the field trial, collection of research literature and consultations with field specialists. The field trial was initiated on April 10, 2023. Details of the methods I used are presented below along with planned future work relating to objectives 2 to 4.
MATERIALS AND METHODS
Trial site
The first experimental field trial began on April 10, 2023 at the University of Florida/IFAS Plant Science Research Education Unit (PSREU) in Citra, Florida. Soil at this site is primarily sandy. Soil samples were submitted to the Soil Testing Laboratory at the University of Florida to characterize fertility and organic matter content. Previous cropping history in this plot included tomato, sorghum, and corn, but most recently remained a weedy fallow.
Preparation of inoculum
RKN M. incognita race 3 was increased on tomato (Solanum lycopersicum L. cv. BHN 589) and maintained in the screenhouse. Eggs were extracted from the infected tomato roots by using a 0.25% (w/v) sodium hypochlorite solution, then captured on a sieve with 25-mm-pore openings. The eggs were allowed to hatch at room temperature using a modified Baermann technique with the eggs placed on a wire mesh lined with moistened Kim wipes inside a Tupperware. Only 24-h-old J2s were used for the experiment.
Plant material and preparation
The pepper varieties/advanced lines used in the current experiment is provided in Table 1. CM334 and Charleston Belle carry the Me3 (=Me7) and N gene, respectively, which confer resistance to M. incognita, M. arenaria, and M. javanica. The breeding line UFRJ107(6)A3 confers resistance to the same three RKN species, but the resistance gene that it carries is currently unknown. Plants were grown under greenhouse conditions in potting medium for 6 wks, then moved into 3” peat pots filled with pasteurized field soil from Citra. The plants were then inoculated with 300 infective-stage juveniles (J2s) of M. incognita.
Table 1. List of pepper (Capsicum annuum) varieties and advanced breeding lines used in the current experiment and expected resistance to root-knot nematode (Meloidogyne spp.) with the genes responsible for resistance, if known are shown in brackets.
| Gentoype | Expected resistance to Meloidogyne spp. |
| CM334 | Resistant (Me3) |
| Charlestone Belle | Resistant (N) |
| Early California Wonder | Susceptible |
| Yolo Wonder L | Susceptible |
| Jimmy Nardello Italian | Susceptible |
| UFRJ107(6)A3 | Resistant (unknown) |
Compost selection and composition
Chicken manure was sourced from BWI. (Espoma Organic Chicken manure, 5-3-2 NPK, 2% water soluble N, 3% water insoluble N, and Ca 8% - based on information from the supplier). Chicken manure samples were sent to UF/IFAS Soil Testing Lab (Gainesville, FL) to determine nutrient composition, pH, NH4, % solids, % moisture, % ash, and organic matter.
Trial design and management
The experiment consists of a split block design with two subplots, each consisting of six treatments and three replicates. Each experimental plot was planted with six replicate pepper plants. Poultry manure treatments were top-dressed on planting beds at a rate of 2.8 t/ha. Plants were grown on beds 2.5 feet wide and 120 feet long (300 ft2), at 1.5 ft. between plant spacing, and 5.5 feet between center to center of each bed (Figure 1 and Figure 2).
The subplots were divided based on preplant application of poultry manure and unamended control. The total amounts (in lb) of N, P, and K, added from the poultry manure per bed were 2.5, 0.66, and 0.83, respectively. To eliminate differences in soil nutrition between compost-treated and untreated subplots, 31.4 grams of 10-10-10 N:P:K granular fertilizer were added around the base of each plant in the untreated subplot. In-season fertigation with Fish Emulsion Fertilizer 5-1-1 N:P:K (Fertilome, Bonham, TX), was applied through drip irrigation lines weekly throughout the production season, with the first injection event starting one week after transplanting. Both subplots were further divided into three equal sections for random assignment of varieties and breeding lines treatments.
Figure 1. Visual representation of field trial design.
Soil sampling and nematode population estimates
A soil core sampler was used to collect a single sample of soil from each block within each row one week after planting to (1) estimate initial populations of plant-parasitic nematodes and (2) identify any potential block effects present in later analyses (i.e., distribution of plant-parasitic nematodes in soil). Soil samples were submitted to the Nematode Assay Laboratory at the University of Florida (Gainesville, FL).
Statistical analysis
The field experiment was designed as a split block design with 2 treatments and 3 replications. Mean separation tests will be done following analysis of variance using Tukey’s multiple range test at p< 0.05.
RESULTS
Soil sampling and nematode population estimates
The initial population of plant-parasitic nematodes (PPNs) varied widely between individual rows and even blocks (Table 1). The highest density species of PPN was ring nematode (Mesocriconema spp.¸Criconemoides spp., and Criconemella spp.) with a mean of 174 per 100 cm3 of soil across all rows and blocks, while the lowest density species of PPN was lance nematode (Hoplolaimus galeatus), with a mean of 0.0833 per 100 cm3 across all rows and blocks. Root-knot nematode (Meloidogyne spp.) had a density of 8 per 100 cm3 of soil across all rows and blocks.
Table 2. Nematode assay report of soil samples collected at preplant stage of current field trial.
|
Nematode typea |
Population (No./100 cc soil) |
|
|
Row 1 Block 1 |
Lesion |
2 |
|
Ring |
111 |
|
|
Root-knot |
3 |
|
|
Row 1 Block 2 |
Ring |
40 |
|
Root-knot |
2 |
|
|
Row 1 Block 3 |
Lesion |
3 |
|
Ring |
150 |
|
|
Root-knot |
17 |
|
|
Row 2 Block 1 |
Lesion |
19 |
|
Ring |
141 |
|
|
Dagger |
2 |
|
|
Root-knot |
3 |
|
|
Row 2 Block 2 |
Sting |
2 |
|
Lesion |
2 |
|
|
Ring |
100 |
|
|
Row 2 Block 3 |
Lesion |
5 |
|
Lance |
1 |
|
|
Ring |
279 |
|
|
Row 3 Block 1 |
Sting |
5 |
|
Lesion |
4 |
|
|
Ring |
200 |
|
|
Root-knot |
22 |
|
|
Row 3 Block 2 |
Sting |
3 |
|
Ring |
81 |
|
|
Row 3 Block 3 |
Lesion |
21 |
|
Ring |
300 |
|
|
Row 4 Block 1 |
Lesion |
1 |
|
Ring |
281 |
|
|
Root-knot |
30 |
|
|
Row 4 Block 2 |
Sting |
5 |
|
Lesion |
20 |
|
|
Ring |
200 |
|
|
Root-knot |
17 |
|
|
Row 4 Block 3 |
Lesion |
19 |
|
Ring |
205 |
|
|
Root-knot |
7 |
aLesion, root-knot, ring, dagger, sting are prevalent hosts for pepper (Capsicum spp.)
Data on vegetative growth will be collected when 50% of plants in the subplots have at least one open flower (i.e., time of 50% flowering). Measurements will be taken on plant height, plant canopy width, stem length, mature leaf length, and mature leaf width for a total of three replications per variety per block. The expected time for collecting this data will be from June-July, depending on weather conditions. Additionally, weeds will be manually removed from the planting beds while also scouting for diseases and pests on a bi-weekly basis. Squash transplants will be prepared to plant in the same bed after harvest for objective three of the current study. The expected time of harvest will be July-August, at which three experimental procedures will be conducted. First, soil samples will be collected to calculate changes in RKN soil populations over time. Second, three varieties per block will be evaluated for galling to rate degree of resistance to RKN under the organic production system and field conditions. Third, roots of the same three varieties per block which were evaluated for galling will be processed to collect root extracts for objective two of the current study.
Deliverables
At least one peer-reviewed publication is expected as the outcome of this research.a
Educational & Outreach Activities
Participation Summary:
An EDIS article will be published as a result of the experiments, which are still in progress. The information provided in the future EDIS article would benefit underserved growers using organic production systems to produce pepper and squash. The first trial begun the first week of April, 2023.