Intercropping chickpea with flax: An alternative sustainable way to manage Ascochyta blight of Chickpea

Objective 1: Select and test chickpea varieties and breeding lines for their compatibility or competitiveness with flax

Objective 1a: Screen chickpea seeds for Ascochyta infestation

Before planting chickpea and flax together to screen their compatibility at EARC and SARC, chickpea seeds need to be screened by a seed health assay. First, 600 seeds of each chickpea cultivar were sterilized in a 1% chlorine solution for 10 min and then rinsed with sterile water. The seeds were then air-dried in a biological cabinet for 30 min and plated on potato dextrose agar for 7 to 10 days with 10 seeds per plate. The plates were examined for the presence of pathogens by viewing the colonies and fruiting bodies at 40X magnification. Finally, the percentage of Ascochyta contamination were calculated as n/N*100, where n is the number of Ascochyta infected seeds and N is the total number of seeds tested.

Damping-off and root rot are diseases of concern in chickpea production and can cause seedling loss. A complex of soilborne pathogen is responsible for damping-off and root rot in the Northern Great Plains. A seed treatment study was carried out for 25 chickpea genotypes in 2020 and 2021. The chickpea seeds with and without fungicide seed treatment (fluxapyroxad, pyraclostrobin, and metalaxyl) were planted at the Eastern Agricultural Research Center in Sidney, MT. Stand counts were recorded three times to calculate area under disease progress curve (AUDPC). Chickpea yields were assessed after harvest.  

Objective 1b: Test chickpea cultivars for compatibility with flax

In this project, sixteen chickpea cultivars were intercropped with one flax cultivar and mono-crop plots were also planted for comparison. Since flax and chickpea had different seed sizes and seeding depths, the chickpea and flax were planted in alternate rows. The experiment was laid out in a randomized complete block design with four replications. The seeding rate of mono-crop chickpea and mono-crop flax was 4 seeds ft^-2 and 68 seeds ft^-2, respectively.  For intercropping, chickpea and flax were planted in alternate rows at 4 seeds ft^-2 and 34 seeds ft^-2 for chickpea and flax, respectively, i.e., one row chickpea was intercropped with one row flax in a 5 × 20 ft plot. The row space between the chickpea and flax was 4.3 in. Each plot consisted of 6 chickpea and 6 flax rows. The mono-cropping chickpea and flax has row spacing of 9 in. 

Chickpea seeds were treated with Obvius Fungicide (BASF Corporation, Research Triangle Park, NC) at 0.18 g a.i. kg^-1 seed and Cruiser 5FS Insecticide (Syngenta Crop Protection, Inc., Greensboro, NC) at a rate of 0.5 g a.i. kg^-1 seed before planting. At planting, it was inoculated with a commercial rhizobia inoculant (Primo GX2 Verdesian Life Sciences, Cary, NC).

The emergence time and plant density will be measured after plant emergence.

A bundle plant sample for determining shoot length, pod number per plant, seed weight, shoot weight, and shoot nitrogen (N) of chickpea and flax was taken by hand cutting the plants from soil surface on August 2, 2021, and August 19, 2022, respectively.

Crops were harvested by a plot combine harvester (Wintersteiger, Salt Lake City, Utah, USA) at maturity on August 17, 2021 and August 31, 2022. After harvesting, the total seed weight was determined from each plot. The flax and chickpea grains were then separated using a screen to determine the yield of each companion crop under intercropping. The chickpea and flax yield data were reported on a dry mass basis.

Seed N concentrations of chickpea and flax were determined by the Pregl-Dumas method on a Perkin Elmer 2400 Series II CHNS/O Elemental Analyzer (PerkinElmer Inc., Waltham, MA). Specifically, 50g chickpea seed sample and 5g flax sample were homogenized into fine powder (<6 mm) by an UDY cyclone sample mill (UDY Corporation, Fort Collins, CO). In the meantime, the moisture content of the ground sample was measured by weighing 1.5 g samples before and following a 48-hr oven-drying at 65 °C (Jones Jr & Case, 1990). Seed protein concentration was calculated by multiplying the total grain N concentration by an N-to-protein conversion factor of 6.25. The protein concentrations reported in this paper are on a dry mass basis.

The land equivalent ration (lER) and net effect ratio (NER) were calculated for assessment of land use efficiency of inter cropping and competitiveness of chickpea cultivars:

The LER is calculated as the sum of the relative yields of intercropped species compared with their respective sole crops (Mead & Willey, 1980) as following (Eq. 1):

LER = Y1/M1 + Y2/M2 = pLER1+pLER2 (Eq. 1)

Where Y1 and Y2 are the yields of species 1 and 2 in the intercropping, M1 and M2 are the yields of species 1 and 2 in the sole cropping, and pLER1 and pLER2 are the partial land equivalent ratios for species 1 and species 2, respectively.

The NER is defined as the ratio of the observed yield of both species to the expected yield that is expressed as a weighted sole crop yields according to the proportions of each crop species in the mixture (Eq. 2) (Cardinale et al., 2007; Loreau & Hector, 2001).

NER = (Y1+Y2)/(p1M1+p2M2) (Eq. 2)

Where Y1, Y2, M1, and M2 are the observed species 1 and species 2 yields in the intercropping and monocropping, respectively. P1 and P2 are the proportions of species 1 and species 2 in the intercropping.

Objective 1c: Configure intercropping spatial distributions for yield and disease suppression

Two chickpea cultivars (CDC Leader and Royal) were planted as monocropping and intercropping with one commonly grown flax cultivar (Glas) at EARC and SARC in alternate row configuration with seeding rate of: 1) sole crop chickpea, 2) sole crop flax, 3) alternate rows planting chickpea (4 seeds ft^-2) with flax (30% recommended rate) planted between the chickpea rows, 4) alternate rows planting chickpea (4 seeds ft^-2) with flax (50% recommended rate) planted between the chickpea rows, 5) alternate rows planting chickpea (4 seeds ft^-2) with flax (70% recommended rate) planted between the chickpea rows. These treatments will impose different degrees of competition levels from flax on chickpea. The plant growth, canopy development, and yield were be determined. Disease incidence and severity were evaluated at different growth stages. 

Objective 2: Study the effect of intercropping on disease severity and dispersal of Ascochyta rabiei

Ascochyta rabiei has two primary methods of spore dispersal in the field. Ascospores from pseudothecia (a sexual round- or flask-shaped fruiting body) are produced on chickpea residue from the previous season and can travel by wind kilometers from the original source. These spores frequently serve as the primary inoculum for disease introduction into a field. Pycnidiospores from pycnidia (an asexual, flask-shaped fruiting body), produced both in chickpea residue and on diseased plant tissues, are spread short distances through water splashing. Pycnidiospore movement accounts for spread within a field and when weather conditions are ideal can result in large disease foci where there are complete crop losses.

Intercropping chickpea and flax has been found to lower disease within the field, however the mechanism for the decrease is still unknown. It has been hypothesized that this decrease may be caused by 1) reduction of long distance movement of ascospores across a field, 2) interruption of short distance movement of pycnidiospores within the canopy, or 3) a change in microclimate within the canopy which decreases disease severity and incidence.

An on-station epidemiological study was conducted at two locations: the MSU Eastern Agricultural Research Center in Sidney, MT in 2022, 2023, and 2024, and the Southern Agricultural Research centers in Huntley, MT in 2022 and 2023. In addition, two on-farm trials with a subset of disease variables were planted at Geraldine and Fallon, MT in 2023.

Objective 2a. Determine if intercropping slows ascospore movement within a field by using spore traps to measure inoculum amounts over time.

Objective 2b. Determine if intercropping reduces the movement of pycnidiospores within the canopy by measuring disease foci spread.

Objective 2c. Understand changes in canopy microclimate between mono- and intercropping designs by measuring factors important for disease development; specifically humidity, temperature, and leaf wetness.

Objective 2d. Measure the impact of fungicide applications in both cropping systems on disease control and seed quality.

A split-plot design was used for both on-station and on-farm trials, incorporating cropping systems and cultivars as main plots and fungicide treatments as subplots. The main plots were randomized within the block and included four replications. Four kabuli-type chickpea cultivars, differing in AB resistance levels, were planted, with two cultivars at each location. The cultivars “CDC Leader", "Royal", "Sawyer", and “CDC Orion” were intercropped with one flax cultivar (Glas) in an alternate-row configuration. Sole chickpea plots served as controls in the on-station trials, while both sole chickpea and sole flax plots were planted as controls in the on-farm trials. “CDC Leader” and “CDC Orion” were released by the Crop Development Center (CDC) at the University of Saskatchewan in Canada and are regarded as moderately resistant cultivars (Ashokkumar et al., 2014; Taran et al., 2011). The cultivars “Sawyer” and “Royal” are identified as moderately susceptible cultivars and were released by the U.S. Department of Agriculture - Agricultural Research Service (USDA-ARS) (https://washingtoncrop.com/documents/Chickpeas/Sawyer.pdf; Vandemark et al., 2019). The main plots measured 36 x 36 ft in the on-station trials and 129 x 350 ft in the on-farm trials. In the on-station trials, chickpeas were planted at the recommended rate of 4 live seeds ft^-², at a depth of 2 in and 9 in apart between rows. In the intercropping system, chickpeas and flax were planted in an alternating-row configuration, with flax sown 6 in from the chickpea rows at a depth of 1.0 in, using 50% of the recommended seeding rate of 68 seeds ft^-². In the on-farm trials, chickpeas were planted at the full recommended rate of 168 kg/ha, while flax was planted at 40% of the recommended rate, which is 22 kg/ha. 

Fungicide treatment applications:

In the on-station trials, each main plot was divided into three subplots, with each subplot receiving a specific fungicide treatment. The treatments consisted of no fungicide, minimal spray, and standard spray applications, which were randomly assigned to each subplot within the main plots and replicated four times. The subplot measured 12 x 36 ft for all on-station trials conducted over the years. The number of applications varied across years and locations based on weather conditions and disease progression for the on-station trials. In the on-farm trials, replications 1 and 2 received two applications, while replications 3 and 4 had the fungicides applied once. To address previously identified pathogen resistance, different modes of action were utilized, excluding Frac 11 fungicides. Trials were evaluated for Ascochyta blight beginning at early flowering and every two weeks thereafter for approximately eight weeks. 

Experimental plot management:

To protect the seeds from seedborne and soilborne pathogens and insects, they were treated with Obvius (fluxapyroxad, pyraclostrobin, and metalaxyl, BASF Corporation, Research Triangle Park, NC) at a rate of 0.18 g a.i./kg seed and Cruiser 5FS insecticide (Thiamethoxam, Syngenta Crop Protection, Inc., Greensboro, NC) at a rate of 0.5 g a.i./kg seed before planting for the on-station trials. During planting, chickpeas were inoculated with commercially available rhizobial inoculants (Primo GX2 Verdesian Life Sciences; Exceed superior legume inoculant garbanzo bean # 4002). The trials were harvested using a combine harvester (Wintersteiger, Salt Lake City, Utah) eight to ten days after applying a desiccant (Gramoxone 3LB, Syngenta Crop Protection, Inc., Greensboro, NC).

Description of the spore traps:

A spore trap adapted from a previously published protocol (Quesada et al., 2018) was used at all four locations. The main body of the spore trap was a large can. The bottom of the can was removed with a can opener, and several holes were drilled and strategically placed to support the assembly. The top bar of the sampler was marked and drilled 1 inch above the can handle holder and measured 3 ¾ inches lengthwise, intersecting to create a central hole. Additional holes for zip tie support were drilled ¼ inch above and below the main body handle holder, with precise measurements to ensure alignment. For the bottom connection, a smaller hole approximately 3 ¾ inches long was made 4 ½ inches from the can handle holders. Galvanized steel wires of varying lengths (7, 6, and 3 inches) were cut and attached to form a sturdy framework. The 6-inch wire featured a fish spinner for mobility, while the 3-inch wire, equipped with alligator clips, served as support for the rods. The wind wheels, made from aluminum can circles, were designed with slits and secured using mini hot glue stick (Gorilla Glue Clear Hot Glue Sticks, 8" x .27") and a corded hot glue gun (Surebonder^® LT-160F Low Temperature Mini Hot Glue Gun, FPC Corp. Wauconda, IL, USA) to ensure stability. They were arranged in a unique rotational pattern on the 6-inch wire to improve airflow. After assembly, the spore trap was coated with green spray paint for easy identification and installed in the field using a metal post (60 in. x 1.2 in. x 1.2 in. Metal U Fence Post, Green). Beginning at flowering, spore traps were placed in plots 2 ft above the canopy both in on-farm and on-station trials to determine the amount of spore movement within the field. 

Preparation of sampling rods:

The injection-molded clear plastic rods (Aerobiology Research Laboratories, Ontario, Canada), measuring 1.59 mm x 1.59 mm x 32 mm, were prepared following a protocol based on Thiessen et al. (2016). First, the plastic sampling rods were washed with a solution of dishwashing detergent and water. They were then soaked in a 10% bleach solution (7.5% sodium hypochlorite) for 15 min, followed by 3 to 4 rinses with sterile water. Afterward, the rods were aseptically air-dried inside a biological safety cabinet. Once dry, a thin layer of Versilube® G600 Series silicone compound (Aerobiology Research Laboratories, Ontario, Canada) was applied using gloved hands. Finally, pairs of coated rods were secured in plumbers' putty (Harvey™, Nebraska, USA) attached to the lid of a sterile 14 ml Falcon tube for convenient handling during downstream use.

Placement of spore traps and collection of roto rod samples:

Pairs of prepared rods were placed in the spore trap about 3 to 4 weeks after planting and removed before applying the first fungicide treatment, which coincided with the appearance of the first AB symptoms in the field. The rods were collected and replaced twice a week for both the on-station trials and the Geraldine location and once a week for the Fallon location. The sampling rods were transported in a 2 ml tube that was labeled and dated. The tubes were stored at -20°C until processing. The starting and ending dates of spore trapping for each location and year are shown in Table 4.6.

DNA extraction from roto rods and identification and quantification of A. rabiei inoculum:

DNA was extracted from the sampling rods using the protocol developed by Thiessen et al. (2016). The amplification of the reactions was carried out using a 20 µl reaction mixture that included 10 µl of Sso Advanced Universal SYBR^® Green Supermix (Bio-Rad Laboratories, Inc.), 250 nM of each primer, 1 µl of the DNA sample, and 7 µl of sterile water. The conditions for qPCR amplification used a 2 Step Amp + Melt protocol consisting of initial denaturation at 95°C for 4 min, followed by 39 cycles at 95°C for 30 sec and 60°C for 30 sec, with a final extension step at 95°C for 10 sec and a melt curve from 65°C to 95°C, increasing by 0.5 °C every 5 sec. The reactions were performed in duplicate, and a standard curve with a tenfold serial dilution ranging from 50 ng/µl to 0.5 pg was run alongside the unknown DNA samples. The qPCR amplicons were visualized and processed as previously described for conventional PCR amplicons. Multiple pairs of primers were used for the amplification reactions.

Measuring the spread of disease foci in Sidney:

The experimental subplots were systematically scouted at the seedling stage (three to four weeks after seeding) to identify the initial appearance of disease foci. Each subplot was inspected in a W shape, focusing on plants displaying disease symptoms (Bogdan, 2018). Once potential disease foci were identified, 3 to 4 areas per subplot were marked with colored flags for easy identification and tracking. Disease progression was monitored weekly over a four-week period by counting the number of plants showing symptoms, both within rows and across adjacent rows, while also measuring the distance of the diseased plants from the foci using a meter stick for the years 2023 and 2024.

Collection of weather data at both canopy and field levels:

Environmental conditions were monitored using sensors strategically placed throughout the experimental sites. A temperature, dew point, and relative humidity sensor (HOBO by Onset RXW-THC-900), along with a leaf wetness sensor (HOBO by Onset RXW-LWA-900), were installed in the subplot receiving the highest fungicide application, resulting in a total of 32 sensors per field. Due to economic constraints, these data were collected exclusively for the on-station trials. Data on wind speed, direction, and precipitation were collected at the field level for both on-station and Fallon trials. The sensors were placed centrally within the field (or in a location with a good satellite signal for Fallon) to ensure optimal data collection. Rainfall data for the Sidney site was obtained from the North Dakota Agricultural Weather Network (NDAWN; https://ndawn.ndsu.nodak.edu/), while similar data for the Huntley site came from the weather network at the Southern Agricultural Research Center, Montana State University (https://www.sarc.montana.edu/). All sensor data was recorded daily in a HOBOnet Wireless Sensor Network (HOBO RX2106-900 MicroRX Station) using a data logger positioned at the center of the field, then sent weekly to the user via email for downstream analysis.

Disease evaluations:

Foliar disease assessment began with the observation of the first symptoms and continued biweekly for about eight weeks. Disease severity was evaluated on 50 plants per plot. The percentage of plant tissue affected was visually measured using a 0-100 percentage rating scale, where 0 to 10 represented no infection to small lesions; 11–20: some stem lesions–minor stem breakage in upper foliage; 21–30: 1–2 branches broken–several girdling stem lesions low down on some branches; 31–40: large basal stem lesions or several branches broken near to main stem; 41–50: half foliage dead or partly severed; 51–60: > more than half foliage dead or dying, young shoots still actively growing from base; 61–70: most foliage dead–some healthy stem tissue with lateral buds; 71–80: most foliage dead, no healthy lateral buds in leaf axils; 81–99: most foliage dead, decreasing areas of living stem tissue; and 100 indicated completely dead plants (Gowen et al., 1989; Zhou et al., 2022b). Disease incidence was recorded as the percentage of plants showing symptoms relative to the total number of plants evaluated. The dates on which the data for disease severity were collected for each location and year are provided in Table 4.5. After pod development, disease symptoms on 50 pods were rated using a 0 to 5 scale from Roger & Tivoli (1996).

Post-harvest data collection:

After harvesting, the seeds were analyzed for yield, moisture content, test weight, protein levels, and oil content. For the intercropped plots, flax and chickpea seeds were separated using a 3.5 mm round screen to determine the yield of each crop, which is reported on a dry mass basis. The grain yield data for chickpeas and flax were calculated based on the weights of cleaned seeds and the area of the harvested subplot after adjusting the moisture content to 13% and 6.5%, respectively. A total of 100 g of chickpeas and 10 g of flaxseed were sub-sampled for measuring oil and protein content. The seeds were processed into a fine powder using a UDY cyclone sample mill (UDY Corporation, Fort Collins, CO). The moisture content of the samples was evaluated using the oven dry method (Jones Jr. & Case, 1990; Reeb et al., 1999; Ahn et al., 2014). The evaluation was performed by weighing 1.5 g portions before and after a drying period in an oven at 65°C for 48 hours (Zhou et al., 2022b). The protein level for both crops was determined by multiplying the total grain nitrogen content by a conversion factor of 6.25 to calculate the N-to-protein ratio, with all protein values expressed on a dry mass basis (Tkachuk, 1977; Krul, 2019; Zhou et al., 2022b). The nitrogen (N) content of both chickpea and flax seeds was analyzed using the Pregl-Dumas method along with a Perkin Elmer 2400 Series II CHNS/O Elemental Analyzer (PerkinElmer Inc., Waltham, MA). The quantification of oil content from flaxseed was processed using a nuclear magnetic resonance (NMR) analyzer (Oxford Instruments Industrial Analysis Group, Abingdon, Oxon). A total of four grams of flax seeds was calibrated against pure flaxseed oil, and the resulting data is expressed as the percentage of oil content based on a dry mass basis (Zhou et al., 2022b). Screening chickpea seeds for A. rabiei contamination levels was conducted using the same conventional seed agar method.

Objective 3. To assess economic benefits of intercropping compared to mono-culture of chickpea and flax.

Economic profit is critical for the sustainability of chickpea production. In this project, we will record all the inputs and outputs for the on-station trials in Objective 1c and Objective 2, as well as on-farm trials in Object 2. In the year 3, an economist will be consulted for economic analysis for different intercropping configurations.