The Northeast Arugula Team (NEAT): Evaluation and Mitigation of Limitations to Profitability for Arugula and Brassica Leafy Green Production

We progressed in our three main research areas this year: Identification of arugula and brassica leafy green bacterial pathogens; 2) Field germplasm evaluation; 3) Greenhouse germplasm evaluations.

Identification of arugula and brassica leafy green bacterial pathogens.

Bacterial disease outbreaks were recognized through NEAT scouting & collection efforts, collections made by an extension personnel, and from growers contacting us. For each plant sample, bacteria were isolated using published methods using KBBC, a semi-selective medium for Pseudomonas and general nutrient agar media. Pure cultures were stored, and they were used in hypersensitivity test on tobacco to determine if isolates are likely to be plant pathogens. Organisms inducing an HR in 24-48 hours were stored. The strains selected for long-term storage are then used in PCR experiments to identify the species of the pathogen. Once the bacteria are identified, we will conduct pathogenicity tests on the plants from which they were isolated (arugula or other brassica leafy green). In the 2023 growing season, we collected isolates from 62 samples from five Pennsylvania arugula growing locations and none from other states. Of these 62 samples, 13 plant pathogenic bacteria were isolated, six of which have been successfully genotyped using multi locus sequence analysis. Six Pseudomonas allivorans isolates or a closely related member of the P. syringae species complex were identified. In March 2024 a Grower Network Webinar was held and we demonstrated the differences between significant bacterial disease of arugula and BLGs for growers and NEAT collaborators. This material was also shared with clinic directors from the Northeast Plant Diagnostics Network (NEPDN). Since spring 2024, 34 samples were collected or received by NEAT members. During this time, nine farms were scouted for bacterial disease outbreaks. Of the 34 samples collected, six have been identified as pathogenic are in the process of being genotyped. In collaborative work with cooperator Dr. Hehe Wang we published an article which highlights a qPCR rapid detection method for Pseudomonas cannabina pv. alisalensis, the causal agent of bacterial blight in arugula and brassica leafy greens. We now utilize this method to rapidly detect bacterial blight in arugula samples. (A qPCR assay for specific detection of Pseudomonas cannabina pv. alisalensis”, Noh et al., 2024 DOI: 10.1094/PDIS-06-24-1217-RE ).

Field Germplasm Evaluation

In fall 2023, a preliminary field trial was established to determine nutrient management, irrigation techniques, and pest management strategies for our farming situation where we had not previously grown brassica leafy greens. We were able to successfully grow commercial arugula cultivars under the parameter recommended to NEAT by our PAC & cooperator Bill Boone. We grew 10 cultivars in randomized plots which we did preliminary evaluations on for bolting and flea beetle damage and scouted regularly for bacterial diseases. The cultivars used included: five Eruca sativa cultivars (‘Astro’, ‘Runaway’, ‘Esmee’, ‘Standard’, ‘Balboa’), four Diplotaxis tenuifolia cultivars (‘Bellezia’, ‘Sylvetta’, ‘Wild’, ‘Dragon’s Fire’), and one cultivar of Diplotaxis erucoides (‘Wasabi’). Arugula was planted in plots at high density (3-5 seeds per inch) and in 36 inch raised beds as recommended to us by farmers from NEAT’s PAC as industry standards. Plots were randomized throughout raised beds and each raised bed within the research field is considered a replicate block. Overhead irrigation was utilized, a 20-10-10 NPK nutrient treatment was applied, and no forms of pest control have been applied. Observations were made twice a week to record bolting and pest damage. Dates were recorded for the first signs of bolting within a plot and once >50% of the plants within a plot have bolted. However, late season plantings, cold weather, & shorter days delayed bolting in most cultivars. Consistent bolting throughout the field was only observed in the ‘Bellezia’, ‘Sylvetta’, ‘Wild’, and ‘Wasabi’ cultivars by the time frost terminated the plantings in December 2023. Additionally, no flea beetle damage was seen in any cultivar in this field. This was likely due to the colder weather and late season start. In 2024, two successful field evaluations were conducted in the early summer and fall seasons. 26 commercially available cultivars of arugula were screened for resistance to cannabina pv. alisalensis. The 26 cultivars evaluated were distributed among three species of arugula: 14 Diplotaxis tenuifolia (‘Aphrodite’, ‘Apollo’, ‘Ares’, ‘Athena’, ‘Bellezia’, ‘Dragon’s Fire’, ‘Eros’, ‘Nemesis’, ‘Poseidon’, ‘Red Streaked’, ‘Sylvetta’, ‘Wild’, ‘Wildfire’, ‘Zeus’), 11 Eruca sativa, (‘Astro’, ‘Balboa’, ‘Esmee’, ‘Green Brigade’, ‘Roquette’, ‘Runaway’, ‘Slow Bolt’, ‘Speedy’, ‘Standard’, ‘Surrey’, ‘Uber’), and 1 Diplotaxis erucoides (‘Wasabi’). Arugula was planted in 5ft plots at high density (3-5 seeds per inch) and in 36-inch-wide raised beds as recommended to us by farmers from NEAT’s PAC as industry standards. Plots are randomized throughout raised beds and each raised bed within the research field is considered a replicate block. Overhead irrigation was utilized, a 20-10-10 NPK nutrient treatment was applied, and no forms of pest control were applied. There were three experimental treatments in each evaluation: a natural plot, one plot inoculated with P. cannabina pv. alisalensis (bacterial blight), and another plot inoculated with X. campestris pv. campestris (black rot). Each treatment was made of 4 replicate blocks, with each cultivar randomly grown in 5ft plots within the blocks (26 plots/block). For the experimental plots, bacteria were grown up in nutrient broth and the plants were spray inoculated at maturity. After bacteria was inoculated, disease severity ratings were recorded 7, 14, and 21 days after inoculation. For both bacteria, disease severity was moderate to high among the E. sativa cultivars, and little to no disease was observed among the Diplotaxis cultivars. Flea beetle predation was moderate to high for all cultivars in the summer trial. The fall trial was planted in September and due to the colder temperatures, no flea beetles or bolting was observed among cultivars until the experiments ended in December. For the bacterial blight experiments, similar results were observed in the fall as to the summer trial, with moderate to high disease observed across the E. sativa cultivars, and low-to severe disease symptoms observed in the Diplotaxis cultivars. For the fall Xanthomonas field experiment, there was significantly less disease post-inoculation than was observed in the summer trial. Low-no disease symptoms were observed in the Diplotaxis plots, and only one plot of the E. sativa cultivars had moderate-severe disease symptoms. The low disease spread throughout the field could have been due to a cold front that moved in two days after inoculating the Xanthomonas plots.

Greenhouse Germplasm Evaluations

In late 2023 we established a protocol for our greenhouse evaluations. Arugula seeds were planted randomly among 6-pack planting inserts with a standard Osmocote fertilizer application. An experimental rep would consist of multiple 6-packs, with each cultivar of arugula represented once in each rep. Initial experiments used the original ten cultivars used in the fall 2023 field trials, and the other cultivars were added for later experiments. Planting was staggered for Eruca & Diplotaxis arugula so that all plants would be sprayed at the same time at their maturity date based on the seed packet information. For the bacterial blight evaluations, P. cannabina pv. alisalensis was grown on KMB agar and suspended in buffer to an OD of 600nm which is approximately 10⁸ CFU/ml. For the black rot evaluations, X. campestris pv. campestris was grown in nutrient broth, pelleted, and resuspended in buffer at approximately 10⁸ CFU/ml. The bacteria were sprayed onto the tops & undersides of arugula foliage, and the arugula was placed in high humidity for 48 hours to create optimal conditions for disease infection. Disease severity was recorded at 7, 14, & 21 days after inoculation. Bacterial blight trials were performed from spring-summer 2024, and black rot trials were performed in fall-winter 2024. In the bacterial blight trials, results were similarly mirrored to the summer and fall field experiments, although there was more variation of disease severity among the E. sativa cultivars. In the black rot trials, disease severity was moderate-high among all arugula cultivars. From these experiments, best & worst performers will be identified and selected to be used in 2025 trials that will also include seeds from the USDA-ARS Germplasm Resources Information Network (GRIN).