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Project Reports

NIFA Report Format

 Project title: CONTROL ALT DELETE: ENHANCING RESILIENCY OF BROCCOLI PRODUCTION BY MITIGATING ALTERNARIA LEAF BLIGHT AND HEAD ROT IN THE EASTERN UNITED STATES Report due date: 15 August 2021
 
Award Number: 2020-51181-32062
 
 
III.  Target Audience
 
Narrative: (Enter narrative)
 
Primary audience of the project is the brassica producers in eastern US. This includes broccoli producers, packers, shippers, agronomists, crop consultants, farm managers, field workers, seed companies, and dealers; and broccoli storage and shipping/transport personnel and companies. Other targets for dissemination include the broader scientific community concerned with the evolution of fungicide resistance and population genomics of plant pathogens.  Targets also include postdocs, graduate students, and undergraduate students who receive training in microbiology, molecular genetics, genomics, and scientific communication.  
 
 
IV.  Products
 
Product type: Select one: Book Chapter; Book; Conference Paper and Presentation; Journal Article; Other; Thesis/Dissertation; Websites
 
Status: Select one: Accepted; Awaiting Publication; Other; Published; Submitted; Under Review
NIFA Support Acknowledged: Yes/No; NIFA Acknowledged? Yes/No
 
 
Conference Presentations 
 
Nieto-Lopez EH (presenting author), Cerritos-Garcia D, Koch RA, Doss T, Dutta B, Petkar A, Langston D, Rideout S, Smart CD, Everhart S. 2021. Investigation into the loss of fungicide efficacy for Alternaria leaf blight and head rot of broccoli and cruciferous crops in the Eastern USA. Poster: American Phytopathological Society North Central Division Meeting, virtual. Status: Other; NIFA Support Acknowleged: Yes; NIFA Acknowledged: Yes.
Petkar, A (presenting author)., Doss, T., Koch, R.A., Ji, P., Everhart, S., and Dutta, B. 2021. Identification and hostrange evaluation of Alternaria species complex affecting broccoli in GA. Poster: American Phytopathological Society North Central Division Meeting, virtual. Status: Other; NIFA Support Acknowleged: Yes; NIFA Acknowledged: Yes.
 
 
V.  Other Products
 
For each product, enter the following information:
 
Product type: Select one: Audio or Video; Data and Research Material; Database; Educational Aids or Curriculum; Evaluation Instruments; Instrument or Equipment; Model; New Germplasm; Other; Physical Collection; Protocol; Software or NetWare; Survey Instrument
 
Description: (Enter 1-3 short sentences should be used to describe what the product is, what activity it resulted from, and whom it benefitted.)
 
Product type: Protocol. “DNA minipreps from fungi: Adapted for Alternaria”. This product is a protocol for a quick and cost-effective method for extracting DNA from our Alternaria isolates that resulted from the molecular identification objective. This work benefitted all PIs and associated personnel involved in this study that are responsible for isolating and identifying Alternaria species.
 
Product type: Protocol. “Alternaria specific primer protocol”. This product is a protocol that describes how to use species-specific diagnostic primers to identify Alternaria isolates and obviating the need for sequencing. This resulted from the molecular identification objective. This work benefitted all PIs and associated personnel involved in this study that are responsible for isolating and identifying Alternaria species.
 
Product type: “Protocol. “Survey protocol for collecting infected samples in commercial broccoli field”. This product provides guidelines for surveying infected broccoli samples (leaves and heads) in commercial broccoli fields.
 
Product type: “Web-page”.  https://alternariabroccoliproject.uga.edu/welcome/ A webpage was created to update the progress on different aspects of the project. 
 
 
VI. Accomplishments and what was accomplished under each objective.
 
Project goal statement: 
Objective 1: Utilize genomics, population genetics, and fungicide resistance profiling to characterize population structure and develop diagnostic tool(s) for Alternaria sp. causing ABHR in broccoli.
 
(i) Survey of broccoli crops for Alternaria sp. 
In GA, eight commercial broccoli fields were surveyed in fall 2020 and spring 2021. Overall, 108 Alternaria spp. were isolated, purified and single-spore cultures were stored at -80oC under further use. Another survey will be conducted in fall 2021. 
In VA, three broccoli farms operated by VA Department of Corrections were scouted for the presence of disease in the fall of 2020.  Plants exhibiting symptoms of Alternaria blight and head rot (AHBR) were found in each of the three locations.  Suspect plant tissues were collected and taken to the diagnostic lab at the Tidewater Agricultural Research and Extension Center (TAREC) for identification.  Plant tissues were biopsied for the presence of pathogens on acidified potato dextrose agar (PDA).  Colonies identified as Alternaria spp. were pure cultured from biopsy plates and stored for species identification. Parker Farms in Oak Grove, VA was sampled both in the fall of 2020 and spring of 2021 for Alternaria sp. Several isolates were obtained from these farms.  In addition, farms in Blacksburg and Wytheville, VA were sampled in the spring of 2021.  Further sampling in the fall of 2021 is planned.
In NY, transplant production occurs in April with plants moving to the field in May. Additionally, there is some later season production with transplants going into the ground in late July or early August for the fall crop. CoPI Smart surveyed transplant production facilities in April and May, 2021, but it was a very dry spring, in fact it was dry through the month of June and no Alternaria was seen. Co-PI Smart will continue to look for broccoli fields with Alternaria, and will collect isolates as soon as there are outbreaks in NY.
 
(iii)    Molecular identification through PCR amplification and sequencing of Internal Transcribed Spacer (ITS): 
 
To determine the identity of the causal agents of ABHR along the Eastern Seaboard and test the hypothesis that a novel Alternaria subspecies is a also a causal agent of ABHR along the Eastern Seaboard, we utilized PCR amplification and multi-locus phylogenetic analysis on a subset of collected isolates from each region (NY, VA and GA). Our results suggest that thus far, only A. brassicicola and A. japonica are responsible for causing ABHR in the target region. Additionally, we identified single nucleotide polymorphisms (SNPs) in several of our phylogenetic loci, including the major allergen (Alt a1) and ATPase, in the subset of our isolates, suggesting there is geographic structuring of A. brassicicola. Second, we verified the efficacy of a previously published set of Alternaria diagnostic primers for rapid identification of each of our isolates, obviating the need for sequencing. Isolates that do not amplify at any of the diagnostic primers are continued to be sequenced at the ITS locus to ensure proper identification.  This work is currently in preparation for a short communication for Plant Disease. Additionally, training in molecular lab techniques and methodologies was provided to the graduate student leading this project.
Utilizing A. brassicicola-specific primers, 108 Alternaria isolates from GA collected in fall 2020 and spring 2021 were assayed and confirmed as A. brassicicola. 
 
(iv)    Pathogenicity and aggressiveness of Alternaria isolates collected
PD Dutta and collaborator Petkar has developed a detached leaf assay to assess aggressiveness of Alternaria spp. in brassicas. One hundred percent of the isolates collected were pathogenic on brassica hosts but aggressiveness varied considerably. Based on this assay, 83.7% of the Alternaria isolates were highly aggressive on broccoli whereas only 10.9 and 4.3% of the isolates were moderately and less-aggressive, respectively. Only, 1.1% of the isolates were nonpathogenic on broccoli. 
 
(v)      Fungicide sensitivity and fungicide resistance screening
During this reporting period, Co-PI Everhart has developed and optimized the protocols that the entire SCRI team will use to measure fungicide sensitivity for all isolates collected over the three years of this project. Aspects Co-PI Everhart successfully optimized included determining the use of a spore suspension in these assays and how to induce sporulation in non-spore producing isolates, as well as what concentrations of azoxystrobin to use to get accurate fungicide sensitivity estimations (through EC50 values).   To this end, we have screened 15 A. brassicicola (five each from NY, VA and GA) and five A. japonica (all from GA) isolates for sensitivity to azoxystrobin. These isolates represent historical collections (isolated prior to the use of QoI fungicides) and new collections. Thus far, we have detected a range in EC50  values, suggesting that some of the isolates are intermediately sensitive to azoxystrobin, but none are resistant. Training in conducting fungicide sensitivity assays was provided to the post grad involved in this project. To screen for fungicide resistance in A. brassicicola isolates, we used previously published primer sets that target the known point mutations in the cytb gene that are known to confer QoI resistance in ascomycete pathogens. None of the three known point mutations that confer QoI resistance are present in the 15 A. brassicicola isolates that we used in the phylogenetic analysis and fungicide sensitivity screening. We are currently in the process of optimizing primers that target the cytb gene in A. japonica so that we can screen those isolates for QoI resistance.
 
(vi)    Population genetics: 
An efficient and cost-effective DNA extraction procedure has been optimized for A. brassicicola and all collected isolates have extracted DNA thus far. In order to identify SNPs in our target Alternaria species, we first need to sequence three to four genomes for each species. Currently, we are in the process of deciding which A. brassicicola isolates to send for the preliminary genome sequencing. 
 
(vii)  Development of diagnostic tools:
None of the three known point mutations that confer QoI resistance are present in the 15 A. brassicicola isolates that we used in the phylogenetic analysis and fungicide sensitivity screening. Before diagnostic tools can be developed to rapidly screen for fungicide resistance, we need to understand the genetic mechanism behind the resistance. To this end, we are continuing to screen Alternaria isolates for the point mutations in the cytB gene. Additionally, we are also generating QoI-resistant A. brassicicola mutants in the lab to verify that we can expect to find the known mutations in the cytB genes in resistant isolates.
 
 
Objective 2: Examine how different sources of inoculum and production practices contribute to ABHR outbreaks and develop management programs that are environmentally and economically sustainable. 
 
(i)     Fungicide programs
Co-PI Smart has completed the conventional and organic fungicide efficacy trials in NY, and are analyzing the data. The identical efficacy trails are being performed in GA and VA. There were 12 treatments in the conventional trial, and nine in the organic trial. Plants were inoculated with a NY isolate of A. brassicicola and disease progression was rapid and severe. Plots were rated twice per week for three weeks. Foliar and head disease severity was rated. 
 
(ii)    Evaluation of irrigation practices
Co-PI Smart (NY) has completed the irrigation practices experiment for 2021. To determine the role of timing of irrigation in disease severity, four overhead irrigation treatments were compared. Treatments were 1) fifteen minutes of overhead irrigation at 7 am; 2) fifteen minutes of overhead irrigation at noon; 3) fifteen minutes of overhead irrigation at 6:30 PM; 4) no additional overhead irrigation. This experiment was started July 12 (plants were inoculated on July 12, and irrigation began on July 13). Between July 12 and when the experiment was completed on August 2, Geneva NY was unusually wet as we received over 6 inches of rain, with rain at some point nearly every day. Plots were rated twice per week for both foliar and head disease severity. Data are currently being analyzed. The trials in GA and VA are currently planted.
 
(iii)  Broccoli cultivar trials for ABHR tolerance: 
Cultivar screen trials have just been planted with 27 cultivars in GA, VA and NY. 
 
(iv)  The effect of nitrogen fertilizer rate:  
NY has completed the fertilizer rate experiment for 2021. Plots were rated twice per week for both foliar and head disease severity. Data are currently being analyzed. The trials are underway in GA and VA, which will be completed by Nov 2021.
 
(v)    Evaluation of inoculum sources (weed and seed)
PD Dutta and postdoctoral research associate Kaur will evaluate the host range of Alternaria spp. on wide range of cruciferae, solanaceous, asteracea and cucurbit weeds commonly found in the broccoli growing regions in GA. Three broccoli farms operated by VA Department of Corrections were scouted for the presence of disease in the fall of 2020.  Brassica weeds later identified as wild radish (Raphanus raphanistrum) were found at one location that exhibited symptoms of Alternaria blight and head rot (AHBR).  Suspect plant tissues were collected and taken to the diagnostic lab at the Tidewater Agricultural Research and Extension Center (TAREC) for identification.  Plant tissues were biopsied for the presence of pathogens on acidified potato dextrose agar (PDA).  Colonies identified as Alternaria spp. were pure cultured from biopsy plates and stored for species identification.
 
(vi) Economic analysis [Colson (Economist, UGA)]
 
Economic Analysis – Activities – Year 1
•       Two survey instruments (broccoli growers, project team / stakeholder advisory panel) were developed and administered to obtain estimates of:
o    The prevalence and severity of Alternaria leaf blight and head rot (ABHR) in broccoli
o   Effectiveness of existing management strategies for ABHR
o    Critical research and management needs of the broccoli industry
 
Economics Analysis – Brief Summary of Findings – Year 1

•       Alternaria leaf blight and head rot (ABHR) are a significant economic issue for the broccoli industry.  Both surveyed groups (broccoli growers and the project team / stakeholder advisory panel) agree in this regard.
o    Growers estimate that on average over the past five years 14% of the broccoli crop in their area has been lost due to ABHR.
o    Nearly 25% of growers report experiencing catastrophic losses of more than 50% of the crop in their area during the past five years, as a direct result of ABHR.
o    On average over the past five years, only 16% of growers report minor losses of less than 5%.
 
•       Among an array of existing management strategies (e.g., different fungicides, planting practices, weed management, irrigation, etc.), all surveyed groups agree that there is no single very effective management strategy for reducing economic losses.
o    Among five currently available fungicides on the market, broccoli growers agreed that none of them are very effective in reducing broccoli losses from ABHR.
o    Rotation of broccoli every 2-3 years with a non-brassica is perceived among growers as the most effective of the strategies for reducing economic losses. o Among 20 different management strategies considered, growers revealed a high level of uncertainty regarding the different strategies’ efficacy.
 
•       The average broccoli grower can be characterized as being:

o    Concerned about ABHR.
o      Moderately familiar with and able to identify ABHR in broccoli.
o    Only moderately equipped in terms of management strategies to reduce economic losses.
o    Uncertain about optimal farm management practices to address ABHR.
What opportunities for training and professional development have the project provided? Narrative: (Enter narrative)
 
(Everhart) This project has thus far provided training for four PhD students, four post-doctoral associates, extension agents and consultants.
 
 
Changes/Problems
 
Narrative: Prior to trial establishment, co-PIs conducting the irrigation experiment felt that changing the irrigation timing from a weekly schedule to a daily schedule to better measure the effect of daily leaf wetness period based on the timing of overhead misting.  Timing treatments were decided to be: 1) once early in the morning; 2) once midday; and 3) once before dark.  The hypothesis is that the effect of leaf wetness provided by overhead irrigation would be greater if leaf wetness occurred at different times of the day as opposed to frequency during the week.