Plant and pathogen materials
A sorghum association mapping population known as the sorghum conversion population (SCP) was provided by Dr. Pat Brown at the University of Illinois (now at UC Davis). It has been described previously and is a collection of diverse lines converted to photoperiod-insensitivity and smaller stature to facilitate the growth and development of the plants in US environments. 510 lines from this population were used in this study although due to bad germination and other quality control issues, not all the lines were used in the analysis of all three traits. Ultimately data from 345 lines were used for the analysis of the chitin response, 472 lines for the flg22 response, and 456 for TLS resistance. B. cookei strain LSLP18 was obtained from Dr. Burt Bluhm at the University of Arkansas.
MAMP response measurement
Two different MAMPs were used in this study flg22, (Genscript catalog# RP19986), and chitin . Sorghum plants were grown in inserts laid on flats filled with soil (33% Sunshine Redi-Earth Pro Growing Mix) in the greenhouse. Plants were watered the day before sample collection to avoid extra leaf moisture on the day of collection.
The lines were randomized and, for logistical reasons, were planted in batches of 60 lines. For each line, three ‘pots’ were planted with two seeds per line. Subsequent batches were planted as soon as the previous batch had been processed until the entire population had been assessed. Two experimental runs were conducted for both MAMPs with genotypes re-randomized in each of the two runs.
ROS assays were carried out as previously described. Briefly, for each line, six seeds were planted in 3 different pots. From the resulting seedlings, three were selected based on uniformity. Seedlings that looked unusual or were significantly taller or shorter than the majority were not used. Four leaf discs of 3 mm diameter were excised from the broadest part of the 4th leaf of three different 15-day old sorghum plants. One disc per leaf from two plants and two discs from one plant, with the second disc becoming the water control (see below). The discs were individually floated on 50 µl H20 in a black 96-well plate, sealed with an aluminum seal to avoid exposure to light, and kept at room temperature overnight. The next morning a reaction solution was made using 2 mg/ml chemiluminescent probe L-012 (Wako, catalog # 120-04891), 2 mg/ml horseradish peroxidase (Type VI-A, Sigma-Aldrich, catalog # P6782), and 100 mg/ml Chitin or 2 μM of Flg22. 50 µl of this reaction solution was added to three of the four wells. The fourth well was a mock control, to which the reaction solution excluding the MAMP was added. Four blank wells containing only water were also included in each plate.
After adding the reaction solution, the luminescence was measured using SynergyTM 2 multi-detection microplate reader (BioTek) every 2 min for 1 hr. The plate reader takes luminescence measurements every 2 min during this 1 h. The sum of all 31 readings was calculated to give the value for each well. The estimated value for the MAMP response for each genotype was calculated as (average luminescence value of the three experimental wells—the mock well value) -minus the average blank well value. The blank well values were consistently close to zero.
Leaf discs of Nicotiana benthamiana, one high responsive sorghum line (SC0003), and one low responsive sorghum line (PI 6069) were also included as controls in each 96-well plate for quality control purposes.
B. cookei inoculum preparation and inoculation
B. cookei inoculum was prepared as described previously. Briefly, sorghum grains were soaked in water for three days, rinsed, scooped into 1L conical flasks and autoclaved for an hour at 15psi and 121 °C. The grains were then inoculated with about 5 ml of macerated mycelia from a fresh culture of B. cookei LSLP18 isolates and left for 2 weeks at room temperature, shaking the flasks every 3 days. After 2 weeks, the fungus infested sorghum grains were air-dried and then stored at 4 °C until field inoculation. The same inoculum was used for the entire trial and made fresh every year. For inoculation, 6–10 infested grains were placed into the whorl of 4–5 week old sorghum plants. The spores produced from these fungi initiated infection in the young sorghum plants within a week.
Seed preparation
Before planting in the field sorghum seed was treated with a fungicide, insecticide, and safener mixture containing ~ 1% Spirato 480 FS fungicide, 4% Sebring 480 FS fungicide, 3% Sorpro 940 ES seed safener. Then the seeds were air-dried for 3 days which provided a thin coating of this mix around the seeds. The safener allowed the use of the herbicide Dual Magnum as a pre-emergence treatment.
Evaluation of Target Leaf Spot resistance
The SCP was planted at the Central Crops Research Station in Clayton, NC on June 14–15 2017 and June 20, 2018 in a randomized complete block design with two experimental replications in each case. Experiments were planted in 1.8 m single rows with a 0.9 m row width using 10 seeds per plot. Two border rows were planted around the periphery of each experiment to prevent edge effects. The experiments were inoculated on July 20, 2017 and July 20, 2018 at which point the sorghum plants were at growth stage 3. Ratings were taken on a one to nine scales , where plants showing no signs of disease were scored as a nine and completely dead plants were scored as one . Two ratings were taken in 2017 and four readings in 2018 starting two weeks after inoculation each year. sAUDPC (standardized area under disease progression curve) was calculated as described previously.
Post time: Apr-01-2021