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, each chickpea cultivar will need to be screened by a seed health assay. First, 600 seeds of each chickpea cultivar will be sterilized in a 1% chlorine solution for 10 min, and then rinsed with sterile water. The seeds will then be 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 will be examined for the presence of pathogens by viewing the colonies and fruiting bodies at 40X magnification. Finally, the percentage of Ascochyta contamination will be calculated as n/N*100, where n is the number of Ascochyta infected seeds and N is the total number of seeds tested.

Objective 1b: Test chickpea cultivars for compatibility with flax

Our preliminary studies have been conducted to test the complete mixed vs. alternate rows intercropping with different rates. In this proposed project, since flax and chickpea have different seed sizes and seeding depths, we’ll plant chickpea and flax in paired rows with a randomized complete block design with 4 replications.  Multiple commercial chickpea varieties commonly grown in Montana plus advanced breeding lines from Montana Pulse Breeding Program will be requested and grown alone and intercropped with a commonly grown flax cultivar in the field to assess their compatibility and yield potential.  Chickpea will be planted at a full recommended rate of 43 seeds per square meter at 5 cm depth with 23 cm row spacing. Flax will be planted at 50% of the 28kg/ha recommended seeding rate at 3cm depth and 7 cm from chickpea row. The plot size will be 1.5m x 6m. The emergence time and plant density will be measured after plant emergence. After plant establishment, five plants in each plot will be tagged to monitor plant growth and development biweekly including plant height, number of internodes, and number of branches on the main stem. A dual visual and infrared camera and a Green Seeker will be used to scan the crop canopy periodically to measure canopy structure and proportion of flax and chickpea canopy cover. At the seed filling stage, two rows by one meter long of above ground biomass will be cut by hand at the ground level to evaluate the biomass yield of chickpea and flax, and seed pods and seed number of chickpea. The plant vigor index of each cultivar will be calculated and competition be assessed. At harvest, chickpea and flax grains will be harvested by a plot combine and separated. The yield of each species will be determined, and competition will be assessed with competitive ratio after calculating the land equivalent ratio using following equations:

Chickpea LER = Chickpea yield (intercropped)/Chickpea yield (monocropped);

Flax LER= Flax yield (intercropped)/Flax yield (monocropped).

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

Completely mixed chickpea and flax with different seeding rates has been tested in our preliminary study. In this project, we’ll select two compatible chickpea cultivars to intercrop with one commonly grown flax cultivar at EARC and SARC. The experiment will be laid out as a randomized complete block design with 4 replications. The plot size will be 3m x 10m. The experimental treatments will include: 1) sole crop chickpea, 2) sole crop flax, 3) alternate rows planting chickpea (43 seeds/m2) at 28 cm row spacing with flax (30% recommended rate) planted between the chickpea rows, 4) alternate rows planting chickpea (43 seeds/m2) at 28 cm row spacing with flax (50% recommended rate) planted between the chickpea rows, 5) paired rows planting chickpea (43 seeds/m2) at 28 cm row spacing with flax (30% recommended rate) planted at 7 cm adjacent to the chickpea row, 6) paired rows planting chickpea (43 seeds/m2) at 28 cm row spacing with flax (50% recommended rate) planted at 7 cm adjacent to the chickpea row. These treatments will impose different degrees of competition levels from flax on chickpea. The plant growth, canopy development, and yield will be determined using the same methodologies as in Objective 1b. Disease incidence and severity will be evaluated using the methodology proposed in

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. In the study described here, we will explore the effects of intercropping on these three variables as well as determine if fungicide use can be decreased in the intercropping system.  

We will conduct an on-station epidemiological study for two years (2022 and 2023) at two locations: the MSU Eastern and Southern Agricultural Research centers in Sidney and Huntley, MT. In addition, we will measure a subset of disease variables on two on-farm trials planted at Karl Mavencamp and Cody Meidinger’s farms in 2023 and 2024.

Based on our preliminary agronomic data, chickpea and flax planted in alternate rows perform better than when planted in a complete mixture; therefore we will plant chickpea both mono-cropped and intercropped with flax in the alternate row configuration. Furthermore, to minimize yield penalty to the chickpea crop, chickpea must be seeded >70% of the recommended sole crop seeding rate. Therefore, in this study we will plant chickpea at the recommended sole crop seeding rate with 28 cm row spacing at 5 cm seeding depth. The flax will be planted between the two chickpea rows at 50% of the recommended sole crop seeding rate, with a seeding depth of 2.5 cm.

For the on-station and on-farm studies, two kabuli type chickpea varieties (Ascochyta blight susceptible and relatively resistant) will be intercropped with one flax variety, with chickpea only plots serving as controls. The experimental design will be a randomized complete block design with 4 replications. Plots on-station will receive overhead irrigation according to standard practices to encourage disease development. Natural inoculum is abundant at the research sites, so no artificial inoculations will be used for this study. Each plot will be 12 m × 30 m. The experiments will be planted in April and harvested in early September each year.

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

Spore trapping has been used to quantify spore dispersal in many crop production systems. We intend to use a modified inexpensive spore trap from a previous published protocol (Quesada et. al. 2018) to measure Ascochyta rabiei spore movement within treatments.

Beginning at flowering, spore traps will be 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. Spore collection slides will be exchanged 1-2 times per week for on-station trials and every two weeks for on-farm trials. Slides will be shipped to the EARC Plant Pathology laboratory in Sidney, MT for spore quantification. Spores will be removed from collection slides and DNA will be extracted using a glass bead disruption and a phenol chloroform method. DNA will be subjected to a real-time PCR reaction using the intergenic spacer (IGS) region specific to A. rabiei as the target amplicon (Akamatsu et al. 2012) and DNA quantified using a previously developed standard curve. In 2020, we tested these protocols in the field and were able to both capture and detect A. rabiei spores. We expect to see the treatment effect on amount of the spores collected and direction of movement after data analysis and mapping.

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

Ascochyta blight is usually first detected at early flowering. It typically appears as necrotic spots with rings of pycnidia that resemble a target. Over time these spots will coalesce, causing significant damage to the leaf and eventually infect the pods leading to seed infestation. Under ideal weather conditions, the pycnidia will exude spores which move by water splashing to adjacent plants. It is hypothesized that flax will form a barrier which limits movement of spores through the canopy.

To test this, we will measure disease movement within the canopy in the on-station experiments using a modified protocol (Kimber et al. 2007). At early flowering, plots will be scouted regularly and when disease is first detected, 3-4 potential disease foci per plot will be marked with a colored flag. The spread of disease from the primary focus will be recorded weekly for 5 weeks by counting the number of surrounding plants showing disease symptoms, both in row and across rows. Starting from the focus, infected plants will be counted within 1 m bands using a pivoting 8 m string with 1 m graduations. Spatial distribution of infected plants will also be recorded on a plot map.

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.

Environment plays an important role in the development of Ascochyta blight. Rapid disease development will occur at temperatures between 15-25°C and previous research has found that a relative humidity (RH) of above 86% was required for spore germination and penetration into the host tissue (Navas-Cortes et al. 1998). Additionally, temperature and humidity can have a significant effect on spore viability.

One hypothesis is that flax changes the microclimate of the chickpea canopy so that it is less than ideal for disease development. To address this, temperature/relative humidity sensors and leaf wetness sensors (Onset^®) – two of each per plot - will be placed in on-station plots to measure canopy environmental variables over time. For both the on-farm and on-station experiments, sensors for measuring ambient temperature, humidity, rainfall, and wind speed/direction will also be included.

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

Fungicides are an important component of integrated pest management of Ascochyta blight. When environmental conditions are right, multiple fungicide applications are needed to control disease. We hypothesize that intercropping will both decrease the amount of disease in a field and decrease the number of required fungicide applications.

To address this, each on-station 12 x 30 m plot will be subdivided into three 12 x 10 m sections which receive different fungicide treatments. We will include a no fungicide, a minimum spray, and a standard spray treatment. The number of sprays will be dependent on weather and common practices. Because of previously identified pathogen resistance, modes of action will be rotated.

On-farm studies will have a similar design, except the fungicide applications for disease management will be determined by the farmer in order to secure their participation. It is anticipated that after the first year of the on-station fungicide study that the cooperating farmers may be willing to use a lower fungicide application number. Twice during the season, plots will be evaluated for disease by rating individual plants in the field by the same methods listed below.

For foliar disease, trials will be evaluated for Ascochyta blight beginning at early flowering and every two weeks thereafter for approximately eight weeks. Fifty plants per section will be rated for percent of plant tissue covered with disease (percent severity). Incidence will be calculated as the percent of plants showing disease symptoms. Later in the season after pod development, disease symptoms on pods will be rated on a modified 0-5 scale (Roger and Tivoli, 1996). After harvest, seed will be evaluated for A. rabiei infestation using the seed health assay described in Objective 1a.