Verticillium Wilt Management: Elucidating Mechanisms of Resistance and Integration of Sustainable Alternatives in Tomato Production Systems

Final Report for GS11-102

Project Type: Graduate Student
Funds awarded in 2011: $9,970.00
Projected End Date: 12/31/2013
Grant Recipient: North Carolina State University
Region: Southern
State: North Carolina
Graduate Student:
Major Professor:
Dr. Frank Louws
NC State University
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Project Information

Summary:

Graftingwith various rootstocksis an IPM tool that can provide protection againstsoilbornediseases,and previous studies have documented the successofgraftingto manage bacterial wilt, Fusarium wilt, Southern stem blight and root knotnematodesin fresh market tomato production in North Carolina. In this threeyear study, multiple rootstocks were evaluatedcompared to standardfumigation methods to assess theirefficacyagainstVerticillium wilt, caused by the soilborne fungus Verticillium dahliae. There is no known resistance to the race 2 form of this pathogen, the most prevalent strain in the mountain production region, but in this thesis we hypothesized vigorous rootstocks may confer tolerance to the pathogen. In the absence ofVerticilliumwilt pressureduringtwo ofthe study years,graftingmethods did not affect plant growth oryield when compared to non-grafted control and fumigation treatments. In the third year and in the presenceof Verticillium wilt, rootstocks OH316 and Maxifort providedthe best control againstthe pathogenwhile stilldelivering similaryields as the non-graftedplants in fumigated plots. Rootstocks OH313 and a Japaneseline ‘Aibou’did nottolerate Verticillium wilt pressure as wellasOH316, Maxifort, and thenon-grafted control, which resulted in lower fruityields. Grafting can play an important role in future IPM programs to reduce losses due to this important pathogen especially in high value tomato crops that can compensate for the extra costs of grafted plants.

Introduction

The purpose of this project is to determine the potential benefits of using grafted tomato plants to manage the disease Verticillium wilt in tomato production. Tomatoes are the fourth most popular fresh vegetable in the USA and North Carolina is one of the top fresh market tomato producers {USDA 2009} and has a farm gate value of $33.7 million to the state economy {NCMarketReady 2008}. Unfortunately, there are many different diseases that limit production. One of the more serious diseases is Verticillium wilt (VW) caused by the pathogen Verticillium dahliae. V. dahliae is a soilborne vascular pathogen that enters the host through the roots and colonizes the xylem tissue through the formation of conidia leading to plant stunting, wilt and ultimately plant death.   In conventional systems, the method of choice for controlling V. dahliae is to use fumigants to reduce the inoculum load in the soil before planting. However, with the phase-out of methyl bromide as a soil fumigant and increased interest in organic production in the state, new methods are needed to manage this serious pathogen. Grafting is a potential viable solution to this problem within an IPM framework. Grafting is used extensively in many parts of the world to manage soilborne diseases and reduce abiotic stress, such as salt tolerance, and to increase yields and fruit quality. A major benefit of grafting is that a grower can produce tomatoes (scion) that meet local or regional market demands but select rootstocks that offer site-specific benefits such as resistance to know pathogens on the farm. A major challenge tomato growers face in the mountain production region is that resistance is available to the race 1 strain of the pathogen but no resistance is known worldwide to the race 2 form of the pathogen, the predominant strain in the mountains. Therefore, many growers can suffer serious losses in yield. In this work, we hypothesized that rootstocks that confer plant growth vigor to the scion may reduce crop losses growers experience due to VW pressure. A series of field experiments and lab experiments were conducted to advance our knowledge about the utility of grafting to manage the race 2 strain of V. dahliae.

Project Objectives:

The objectives of this work were to:

1) Evaluate the utility of rootstocks as a viable mechanism for growers to manage Verticillium wilt in fumigated and non-fumigated soils.

2) Advance our knowledge about mechanisms of host resistance through intensive field sampling, PCR analysis and greenhouse assays;

3) Communicate results to growers and other clientele with recommendations of designing integrated and sustainable tomato production systems.

Cooperators

Click linked name(s) to expand
  • Meagan Iott

Research

Materials and methods:

Grafted transplants were produced using the tube grafting method at the Phytotron facilities on the North Carolina State University campus. ‘Mountain Fresh’, MF, was used as the universal scion and grafted onto all rootstock selections and was also used as the non-grafted susceptible control. A self-grafted control was not included since multiple studies have documented that self-grafted and non-grafted controls behave similarly (Rivard and Louws 2008). The experiments were conducted for three consecutive years with selected rootstock (Table 1). Five different rootstocks were grafted to one universal scion (Table 1) and were evaluated at two field sites in Western North Carolina on land with a history of severe Verticillium wilt (VW) pressure. One field site was located at the Mountain Horticulture Crop Research Station (MHCRS) in Mills River, NC while the other was located on a grower’s field in Haywood County (HC). The experimental design had 4 replications and was arranged as a split plot with 4 rootstocks and the non-grafted control randomly assigned as subplots within main plots of fumigation or no fumigation treatments. Parameters measured included weekly plant height, biweekly plant biomass, plant vigor, yield and Verticillium wilt incidence and severity. Two lines from Ohio State University, ‘OH313’ and ‘OH316’, were utilized due to their superior performance in previous regional trials (David Francis, personal communication). An Asahi line ‘‘Aibou’’ was selected as it was a new rootstock that showed promise in Asia experiments against diverse V. dahliae populations. ‘Maxifort’ (MX) and ‘RST-DP106’ (DP106) were included based on prevalence of use in other research efforts (McAvoy et al. 2012; Rivard et al. 2012). DP106 confers southern bacterial wilt resistance and MX confers vigor.

McAvoy, T., Freeman, J. H., Rideout, S. L., Olson, S. M., and Paret, M. L. 2012. Evaluation of grafting using hybrid rootstocks for management of bacterial wilt in field tomato production. Hortscience 47:621-625.

 Rivard, C. L., and Louws, F. J. 2008. Grafting to manage soilborne diseases in heirloom tomato production. Hortscience 43:2104-2111.

Rivard, C. L., O’Connell, S., Peet, M. M., Welker, R. M., and Louws, F. J. 2012. Grafting tomato to manage bacterial wilt caused by Ralstonia solanacearum in the Southeastern United States. Plant Disease 96:973-978.

Research results and discussion:

Objective 1: In 2010 and in 2011, no measurable VW pressure occurred, despite planting on land with a long history of VW pressure. These experiments were done on land at the MHCRS that experienced recent flooding and it is believed the flooding substantially reduced the inoculum load. In 2012, the experiment was repeated at the two sites and the HC site had high VW pressure whereas the MHCRS did not.

Plant height, biomass and vigor: We found plant heights were not affected by fumigation or rootstock treatments in any of the experiments, suggesting this is not a productive parameter to assess rootstock effects. Rootstocks impacted plant dry weight in 2010 but not in the other years. Maxifort tended to increase overall plant growth (Table 2). Fumigation treatment effects were only observed in 2012 where there was a significant increase in plant biomass of fumigated treatments over the non-fumigated treatments at both experimental sites (Table 3).   Rootstock and fumigation treatment effects and their interactions were significant at the HC site in 2012 for plant vigor (Table 4), but not in other years or sites. At the HC site, MX and OH316 tended to have the highest vigor ratings (Table 4). The interaction effects were particularly interesting: MX, the overall most vigorous plants, and OH313, the overall least vigorous plants were not impacted by fumigant treatment. In contrast, all other rootstocks had low vigor in non-fumigated plots and significantly greater vigor in fumigated plots (Table 4).

Disease Incidence: No disease pressure was observed at the MHCRS field site in 2010 and 2011. VW incidence and severity was observed at both field sites in 2012. Rootstock and fumigation treatments significantly affected VW pressure at the HC field site (Figure 1), but not at MHCRS field site (data not shown). At HC, OH313 and V2 had the most VW incidence observed and MX and OH316 had the least. MF had statistically higher AUDPC values of VW incidence than MX and OH316 but lower than OH313, V2, and DP106. Severity of VW associated lesions on MF scions was not affected by rootstock treatments (P = 0.11; Figure 1). Interestingly, fumigation did not significantly decrease incidence (AUDPC values: Fumigation – 991; Non-fumigation – 1224) but did decrease severity AUDPC values (Fumigation – 1750; Non-fumigation – 2344; P=0.047). Interactions were not significant.

Harvest: Rootstock and fumigation treatment effects were not observed in 2010 or 2011 (data not shown). Rootstock and fumigation effects were observed in 2012 at both sites and interactions between rootstocks and fumigants were not significant. At the HC site, OH313 and V2 yielded the least marketable fruit weight when compared with all other rootstocks (Figure 2A). Fumigation treatments also increased marketable fruit weight (Figure 2B). At MHCRS, MX had lower marketable fruit weight than OH313 and V2 (Figure 3A). The MX treatment sustained plant injury and/or poor grafting compatibility at the MSHRS site in 2012, resulting in low yield. Fumigation increased marketable fruit weight overall (Figure 3B). There was a strong correlation at HC between harvest and vigor data when comparing the average marketable fruit weight and the average vigor of plants (Table 13). No significant correlations occurred between harvest and vigor MHCRS data (R2=0.71 P-value=0.03).

Objective 2: Verticillium dahliae verification- Pure cultures obtained from symptomatic plant tissue were identified as V. dahliae based on plate morphology and sequencing of the ITS region. A real-time PCR assay was verified and optimized. One primer set developed by Atallah, et al (2007) amplified Fusarium oxysporum DNA in our hands when both Fusarium oxysporum and V. dahliae DNA were used to test the primers in separate reactions. The amplification was seen when a 1% agarose gel was run. The second primer set from Pasche, et. al, (2013), successfully amplified V. dahliae from plant isolates while not amplifying Fusarium oxysporum. Real-time qPCR protocols were successfully optimized with the second set of primers and protocols to produce amplification and Ct values ranging from 24-33 with the extracted DNA from pure cultures of plant isolates (Figure 4).

Objective 3: Grafting and IPM training workshops were conducted at various times in 2011 and 2012. All workshops focused on educating sustainable integrated pest management strategies (IPM) in regards to diseases on fresh market tomatoes in North Carolina. The majority of workshops consisted of two parts: lecture and hands-on. During the lecture portion of the workshop, a series of PowerPoint presentations were prepared to enable growers and agents to identifying the major diseases of tomato, implement IPM-based solutions, and to learn the methods and benefits of grafting. Subsequently, participants conducted hands-on grafting using the tube-graft method and were provided experience, information and resources to be able to conduct grafting on their farm.

Atallah, Z. K., Bae, J., Jansky, S. H., Rouse, D. I., and Stevenson, W. R. 2007. Multiplex real-time quantitative PCR to detect and quantify Verticillium dahliae colonization in potato lines that differ in response to Verticillium wilt. Phytopathology 97:865-872.

Pasche, J. S., Mallik, I., Anderson, N. R., and Gudmestad, N. C. 2013. Development and validation of a real-time pcr assay for the quantification of Verticillium dahliae in potato. Plant Disease 97:608-618.

Participation Summary

Educational & Outreach Activities

Participation Summary:

Education/outreach description:

Meagan Iott’s Master of Science thesis

Throughout this project, various grafting workshops and one field day were conducted for the benefit of the public. Each workshop consisted of a lecture and hands-on portion. Each workshop provided lessons in soilborne pathogens, various control tactics, and step by step instructions on the ‘tube’ grafting method. The hands-on portion provided attendees with first account training in how to perform the grafting method. The field day occurred at the Mountain Horticulture Crop Research and Extension station during the year with disease absence. The field day consisted of demonstrating grafting in a field setting and introduced an audience of 150 people or more to the prospects of grafting as an IPM tool that could enhance sustainability of tomato production systems. This work complemented a larger research and extension program throughout the Southeast.

Project Outcomes

Project outcomes:

Low VW pressure in 2 of the 3 years limited our capacity to determine the utility of grafting as an IPM tactic to manage this important disease. In the absence of disease, the rootstocks did not offer a yield advantage. Under conditions of high VW pressure (HC in 2012) vigorous rootstocks such as ‘Maxifort’ and ‘OH316’ can reduce disease incidence. In complimentary experiments across multiple farms and not related to this thesis, ‘Maxifort’ has shown increased yield and decreased VW severity, but not decreased incidence. Thus grafting with vigorous rootstock could offer an advantage to growers experiencing VW problems but may not be economical in the absence of disease pressure. Molecular tools were optimized to allow additional work on the mechanisms of resistance. In particular, it would be productive to learn if rootstocks confer a form of tolerance (i.e. allow the pathogen to fully colonize the tissue but limit symptom development) or if there is low level of resistance. Many growers and end users were trained to graft their own plants and gained knowledge about the potential utility of grafting for their operations.

Farmer Adoption

While grafting has not had an explosive adoption by growers in North Carolina, more growers are using the method as an additional tool to control disease. Instead of fumigating all their fields, some growers are using grafted plants in fields that they do not fumigate. While the number of growers adopting the method is low, the number of growers being educated and made aware of the concept has grown exponentially. For each workshop, up to 30 different growers were made aware of the new tool and during field days, more than 150 growers and other stakeholders were introduced to grafting. While most of the growers are still learning about the method, they are actively asking questions about specific techniques to try to see if they could incorporate the new tool into their management plans. There are several specific recommendations that arise from this work to help growers successfully adopt grafting. First, growers must diagnose the pathogens that affect their tomato crop. The overall program has shown good efficacy against a range of soilborne pathogens including major fungal, bacterial and nematode problems. Grafting is best used for soilborne pathogens. Different rootstocks are better equipped to resist or tolerate different soilborne pathogens. Growers need to research which rootstocks would work best in their fields to help control the pathogens present. The second recommendation is to practice the grafting method in small amounts. Grafting is an art form, though not difficult it does take some practice. For example, growers must use proper sanitation practices to prevent spread of diseases and pay careful attention to managing the healing chamber.

Recommendations:

Areas needing additional study

Future areas of research are needed to further study the mechanism resistance/tolerance in the rootstocks. The real-time PCR protocols found through this research need to be expanded to include diseased plant tissue in order to help shed light on how the grafting method invokes tolerance/resistance to soilborne pathogens. Future demonstrations and training are needed on farm sites for the grafting and healing process in small quantities. On-site training will ensure grafting success while allowing the grower to complete the process themselves.

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.