Project Reports

Alternaria Broccoli SAP meeting report 2023

Variety Trial present SAP 2023

Kate Cassity-Duffey and team of the Plant Path department in Athens, Ga

Objective 1: survey, speciation, and host-pathogenicity

By: Chris smart and Team of the Plant Path department in New York

SAP (Stakeholder Advisory Panel) Meeting 2023

Control ALT Delete: Enhancing resiliency of broccoli production by mitigating Alternaria leaf blight and head rot in the eastern United States

Nieto-Lopez EH (presenting author), Cerritos-Garcia D, Koch RA, Doss T, Dutta B, Petkar A, Langston D, Rideout S, Smart CD, Everhart S. 2023

SAP (Stakeholder Advisory Panel) Meeting 2022

Control ALT Delete: Enhancing resiliency of broccoli production by mitigating Alternaria leaf blight and head rot in the eastern United States

Nieto-Lopez EH (presenting author), Cerritos-Garcia D, Koch RA, Doss T, Dutta B, Petkar A, Langston D, Rideout S, Smart CD, Everhart S. 2022

Pictures of Alternaria leaf blight and head rot

Identification of Alternaria leaf spot resistant components in Broccoli

Dr. Sunil Gangurde

Seed-to-Seedling Transmission of Alternaria brassiciola in broccoli

Dr. Navjot Kaur

Project Report Year 2

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 

Conference presentation 

Hirut B, Day, CTC, Smart CD. (2022) Improving control strategies for Alternaria blight and head rot. Presentation at Plant Health 2022 Pittsburgh, Pennsylvania, August 6-10, 2022 

Status: accepted, NIFA Support Acknowledged: Yes 

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 Acknowledged: Yes; NIFA Acknowledged: Yes. 

Species-dependent sensitivity to azoxystrobin observed among causal agents of Alternaria leaf blight and head rot in brassica crops in the eastern US. Poster: American Phytopathological Society Annual Meeting 2022 at Pittsburgh, PA. Status: Accepted; NIFA Support Acknowledged: Yes; NIFA Acknowledged: Yes. 

Petkar, A., Doss, T., Patel, K., Torrance, T., and Dutta, B. 2022. Fungicide sensitivity of Alternaria brassicicola affecting broccoli in Georgia. Poster: Annual Meeting of National Institute of Antimicrobial Resistance Research and Education. May 16, Lincoln, Nebraska. Status: Accepted; NIFA Support Acknowledged: Yes; NIFA Acknowledged: Yes. 

Cassity-Duffey, K., T. Coolong, and B. Dutta. Cultivar trials for Alternaria leaf blight and head rot in  

broccoli. ASHS. July 31, 2022. Chicago, Ill. (Poster). Status: Accepted; NIFA Support Acknowledged: Yes; NIFA Acknowledged: Yes. 

Kaur, N., and Dutta, B. 2022. Seed-to-seedling transmission of Alternaria brassicicola in broccoli.Poster: American Phytopathological Society Annual Meeting 2022 at Pittsburgh, PA. Status: Accepted; NIFA Support Acknowledged: Yes; NIFA Acknowledged: Yes. 

Petkar, A., Doss, T., Patel, K., Torrance, T., and Dutta, B. 2022. Host range, aggressiveness and fungicide sensitivity of Alternaria brassicicola affecting broccoli in Georgia. Poster: American Phytopathological Society Annual Meeting 2022 at Pittsburgh, PA. Status: Accepted; NIFA Support Acknowledged: Yes; NIFA Acknowledged: Yes. 

Webinar: Dutta, B. Update on the national Alternaria project in broccoli. Great Lakes vegetable producers network weekly roundtable, Sep 1, 2021. (Webinar; invited) 

Journal Article 

Nieto-Lopez EH, Cerritos Garcia D, Koch Bach RA, Petkar A, Smart C, Hoepting C, Langston D, Rideout S, Dutta B, Everhart SE. 2022. Species identification and fungicide sensitivity of fungi causing Alternaria Leaf Blight and Head Rot in cole crops across the Eastern U.S.  Status: Accepted for publication with Plant Disease Journal. NIFA Support Acknowledged: Yes; NIFA Acknowledged: Yes. 

Cassity-Duffey, K, T. Coolong, and B. Dutta. 2022. Evaluation of the effect of timing of simulated leaf wetness on disease severity in Alternaria leaf blight and head rot in broccoli, Oconee County, GA 2021. Plant Disease Management Report. 16:V153. Support Acknowledged: Yes; NIFA Acknowledged: Yes. 

Kaur, N., and Dutta, B. 2022. Evaluation of fungicide programs for the management of Alternaria leaf blight and head rot in broccoli in Tift County, Georgia, 2021. PDMR 16:V169. 

Dutta, B., and Kaur, N. 2022. Evaluation of organic fungicides for the management of Alternaria leaf blight and head rot in broccoli in Tift County, Georgia, 2021. PDMR 16:V130 

Dutta, B. and Petkar, A. 2021. Management of Alternaria blight and head rot in brassica. Vegetable and Specialty Crop News, June 2021. 

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. 

A webpage (https://alternariabroccoliproject.uga.edu/) related to the project was created and has been regularly updated for the information related to the project for the stakeholders. 

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. 

Survey of broccoli crops for Alternaria sp.  

In GA, a total of 163 isolates were collected from ten commercial broccoli fields in 2021-2022 season in Georgia from three predominant broccoli growing counties (Colquitt, Grady, and Tift Counties). In NY, over a dozen broccoli fields were surveyed, and four fields were identified that had Alternaria outbreaks. Alternaria isolates (n=119) were collected from four fields in four counties across NY including Niagara, Orleans, Ontario, and Broome Counties. In VA, over 160 Alternaria isolates were collected over 2021-2022 from different brassica species and weeds.  

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 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. Surprisingly, A. japonica was only identified from the isolates that were collected in Georgia. In all three states, more than 90% of the isolates were identified as A. brassicicola. 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.  

Pathogenicity and aggressiveness of Alternaria isolates collected 

Alternaria isolates from GA were tested for their pathogenicity and aggressiveness on detach leaves of broccoli, cabbage, kale and collards.  These isolates were highly aggressive on broccoli (65%), cabbage (46%) and collard (44%) than on kale (8%). In NY, all 119 newly collected isolates were tested for pathogenicity on detached leaves of broccoli, cabbage, cauliflower, kale and collard crops.  Each isolate x crop was replicated three times. Percent disease on each leaf was assessed five days post-inoculation. Broccoli and collards were the two plants most susceptible to all A. brassicicola isolates while cabbage and cauliflower were least susceptible to all isolates. Significant variation among isolates was observed in aggressiveness towards the crops, and the most aggressive isolates were found to be most aggressive on all crops while least aggressive isolates were found to be least aggressive on all crops. In VA, Alternaria isolates are being tested for their pathogenicity and aggressiveness on broccoli, cabbage, collard and kale. 

(v) Fungicide sensitivity and fungicide resistance screening 

In GA, conidial germination assay for a sub-set of isolates on azoxystrobin-amended agar medium indicated 100% of the isolates (n=145) were sensitive at both concentrations (10 and 100 µg/ml). Fungicide sensitivity assay for A. japonica is underway. In addition, Co-PI Everhart have screened 15 A. brassicicola (five each from NY, VA and GA) and seven A. japonica (all from Georgia) 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. We are currently developing a microplate fungicide sensitivity assay based on colorimetric changes of the dye. This high throughput assay will enable us to screen multiple isolates at a time and get the results in less time. 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. 

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 already sequenced six A. brassicicola genomes, and assembled and annotated them. We are currently in the process of mapping the raw reads back to a JGI-generated reference to call SNPs and create a pipeline for understanding the genetic diversity.  

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:  

In GA, six Organic Material Review Institute (OMRI)-listed fungicides were evaluated for the management of Alternaria leaf blight and head rot in broccoli (cv. Emerald Crown) in Tift County. Final disease severity was observed on Oct 20, 2021 with significantly higher foliar severity in all treatments and control plots except for Fontelis (conventional fungicide control). By Nov 1, 2021, disease progressed gradually and none of the treatments including the non-treated check displayed effective disease control. AUDPC values were significantly higher for Double Nickel compared to other treatments and the non-treated check. Furthermore, we also evaluated 11 conventional fungicides for the management of Alternaria leaf blight and head rot in broccoli in Tift County. Based on final disease assessment, disease severity was significantly higher for Switch, Quadris and non-treated check compared to Fontelis, Miravis Prime, Topguard EQ, Bravo Weather Stik, Luna sensation and Priaxor. AUDPC values followed the similar trend as non-treated check, Quadris and Switch had significantly higher values than Fontelis, Miravis Prime, and Topguard EQ.  

In NY, during the 2021 season, conventional fungicides were screened: 12 treatments  were applied four times starting just prior to artificially inoculating with A brassicicola. Cultivar emerald crown broccoli was used and each treatment was replicated four times in a randomized complete block design. All conventional fungicides significantly reduced the disease severity compared with the non-treated control.  

In NY, OMRI-listed fungicides were also screened in a separate experiment. A total of 9 treatments including 6 OMRI-listed fungicides, 2 conventional fungicides and one non-sprayed control were compared for Alternaria leaf blight and head rot. Among the OMRI-listed fungicides, broccoli plants treated with OSO 5%SC had the lowest disease severity. 

 (ii) Evaluation of irrigation practices:  

In GA, an irrigation timing trial was conducted in the Athens location using Emerald Crown as the variety.  All plants received drip irrigation and then four irrigation treatments were implemented: no overhead, overhead irrigation at 6 AM, 12 Noon, and 6 PM.  No significant differences were determined in total yield between misting treatments, but significant differences were determined in the percent disease incidence, number of heads harvested per plot, and marketable heads from each plot.  Overall, there was significantly less disease in the no misting and 6 AM misting treatments compared to treatments that received misting during the day and this was reflected in the marketable heads from each plot. Even with high disease pressure, 67% of heads harvested from the drip irrigated plot were deemed marketable 

In NY, in order to determine the role of timing of irrigation in disease severity, four overhead irrigation treatments were compared. Treatments were 1) 15 minutes of overhead irrigation at 7 AM; 2) 15 minutes of overhead irrigation at Noon; 3) 15 minutes of overhead irrigation at 6:30 PM; 4) no additional overhead irrigation. No significant difference was observed among the treatments. In VA, we conducted a trial evaluating drip vs. overhead irrigation and the effect of leaf wetness period on disease incidence and severity. We did not observe differences between treatments in this trial. We are currently planning on conducting another irrigation trial in Fall of 2022. 

(iii) Broccoli cultivar trials for ABHR tolerance:  

In GA, varieties (n=26) were screened for resistance to A. brassicicola in Athens and Tifton, GA in Fall 2021.  Varieties with low disease in each location included: Marathon, Belstar, Vallejo, and Emerald Pride.  Varieties with low resistance and high disease included Eastern Crown, Mellinium and Asteroid.  

In NY, a field experiment was conducted to determine response of 27 commercial broccoli cultivars to disease. The six-weeks old seedlings were then transplanted mechanically at Research South farm in Cornell Agricultural Research Experiment Station, Geneva, NY. Cultivars were replicated four times using randomized complete block design. Significant difference was observed in susceptibility among the broccoli cultivars with ‘BC 1611’, ‘Montebello’, and ‘Astroid’ being the least susceptible and ‘Green Magic’, ‘Emerald Star’ and ‘Lieutenant’ the most susceptible. A weak negative correlation was observed between AUDPC value and heading days after transplant (-0.24); the negative correlation indicating the earlier the heading date, the more was the disease percentage. In VA, Co-PD Langston and Co-PI Rideout are currently assessing and harvesting a 26 cultivar broccoli trial that has moderate/severe disease pressure. 

(iv) The effect of nitrogen fertilizer rate:   

In GA, a nitrogen fertilizer rate trial was conducted in Tifton and Athens locations using Emerald Crown as the standard variety.  Five levels of nitrogen fertilizer were applied ranging from 50% to 150% of recommended rates for each locationNo significant differences between fertilizer rate and disease were found.  However, higher yields were obtained in the Athens location. 

In NY, five treatments were compared to determine the effect of nitrogen fertility for severity of the disease. The treatments were 50, 75, 100, 125 and 150% of locally recommended N treatment. Half of the recommended fertilizer was applied at planting for the entire plot of the trial. For leaf AUDPC, plots fertilized with 100% N had significantly lower value compared to 50% N treated plots.  

In VA, Co-PD Langston and Co-PI Rideout conducted two trials determine the effect of nitrogen fertility on disease severity.  In the Fall 2021 trial, no significant differences among fertility treatments (50, 75, 100, 125 and 150% of locally recommended N treatment) were observed, but the data did trend towards lower rates of nitrogen having higher disease levels as observed in NY.  

(v) Evaluation of inoculum sources (weed and seed) 

We determined the ability of seed-to-seedling transmission of A. brassicicola in broccoli using artificial infestation of seeds under in-vitro conditions. Seeds (cv. Emerald Crown) were inoculated via vacuum infiltration of conidial suspension (1 × 105 spores/ml). Based on our results, seed-to-seedling transmission of A. brassicicola in broccoli can occur as 100% of the inoculated seedlot transmitted the pathogen indicating seeds could be potential source of inoculum. 

Furthermore, we also explored the effect of the seed inoculum density of A. brassicicola on seed-to-seedling transmission in broccoli. For this experiment, we tested 6 different treatment levels with varying inoculum density including control. Artificial infested seedlots were generated via vacuum infiltration method and SGO assay was conducted as described above. The treatments included: T1 = Control, T2 = 1 × 101, T3 = 1 × 102, T4 = 1 × 103, T5 = 1 × 104, T6 =1 × 105 conidia/ml. Seed germination was impacted and significantly reduced at inoculum level of ≥104 conidia/ml in all the seedlots as compared to the control lot. Consistent seedling transmission occurred at inoculum level of ≥104 conidia/ml in the artificially infested seedlots. We also determined if the difference in the aggressiveness of the A. brassicicola can impact the seed germination and seedling transmission in broccoli. Our study showed that regardless of the variation in the aggressiveness of the A. brassicicola isolates, seed germination and seedling transmission was impacted. We also evaluated commercial seedlots for natural infestation of broccoli seeds with Alternaria. We recovered 73 isolates from 100 commercial broccoli seedlots, which was further confirmed as Alternaria spp.  

In terms of weeds as a source of inoculum, we have collected and isolated Alternaria spp. from cutleaf evening primrose, nutsedge and wild brassica. An isolate found in nutsedge tested positive for A. brassicicola. Currently, more studies are being conducted to verify the other isolates pathogenicity on brassicas species.  

(vi) Economic analysis [Colson (Economist, UGA)] 

Co-PI Colson is currently conducting economic analysis on field trials conducted in GA, NY and VA for 2021 and 2022 seasons. 

What opportunities for training and professional development have the project provided? 

Narrative: (Enter narrative) 

In NY, technicians and postdoc have interacted with colleagues at other universities during quarterly meetings that are part of the grant, expanding their network and skillset. Three high school students worked on the project over the summer as part of an internship program. This has provided them with the experience of working on a plant pathology project that has an impact for broccoli growers. They really enjoyed seeing all the different broccoli cultivars. Hirut Betaw, the postdoc on the project, had the opportunity to present her work at the annual meeting of the American Phytopathological Society in August 2022. Co-PIs Smart and Hoepting also present the work at grower meetings across NY. This project has thus far provided training for two PhD students, one of whom presented his research at a scientific meeting this summer. Another PhD student submitted an article to Plant Disease Journal as a short communication and it was accepted for publication with minor revisions and will be presenting a poster at American Phytopathological Society annual conference 2022. Also involved is a postdoc, Rachel Koch, who has mentored a graduate student on this work thus far. 

Changes/Problems 

Narrative: Changes/Problems 

Narrative: The greatest challenge has been moving to Blacksburg and dealing with the remnants of COVID-19 safeguards that have slowed this process.  Spring 2022 trials evaluating irrigation and fertility were damaged by groundhog feeding.  We plan on conducting trials in Fall 2022 in a field that has not shown groundhog feeding damage in previous years. The researchers were unaware that groundhogs would feed on broccoli until the Spring 2022 irrigation and fertility trials. 

Project report Year 1

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.