TITLE: In vitro co-culture system using a fiber-supported liquid approach
AUTHORS: Alejandro Calle, Jeffrey Adelberg, Guido Schnabel, Jacqueline Naylor-Adelberg, Jhulia Gelain, Yeter Karakoc, Jared Weaver, Christopher Saski, Ksenija Gasic

[DESCRIPTION]
In vitro co-culture techniques that allow the growth of plants and pathogens under controlled environmental conditions are being used to re-create host plant infection. These approaches reduce infection times, promote reproducibility, and enable a rapid evaluation of plant-pathogen interactions. As a result, these systems have become essential in breeding programs aimed at developing plant resistance to diseases. In this study, we developed and validated an in vitro co-culture system to investigate the Armillaria root rot (ARR) affecting Prunus spp. This disease, caused by fungi Armillaria spp. and Desarmillaria caespitosa, poses
a severe threat to the stone and nut fruit industry due to the susceptibility of most commercial rootstocks to infection and the lack of effective management options for its control. The system consists of a fiber-supported liquid approach in sterile plastic vessels that allows a fast and reproducible fungal infection under controlled environmental conditions. The floor of the vessels was covered with a polyester-fiber matte and a germination paper that served as an interface between the mycelia and the plant roots. The vessels were subjected to inoculation with Armillaria mellea and D. caespitosa, and three Prunus genotypes (‘Guardian‱’, ‘MP-29’, and Prunus cerasifera ‘14-4’) were co-cultured with both fungi. Disease progression and plant and fungal biomass were monitored during co-culture. The presented in vitro co-culture approach facilitates the concurrent growth of Armillaria/Desarmillaria spp. and Prunus spp., excluding most of the limitations associated with greenhouses and field experiments. This system provides consistent and reproducible conditions for investigating a prominent plant disease affecting Prunus spp. 

[STEPS]
SECTION: Establishment of Plant Cultures
1. Establishment of plant cultures from dormant shoots
SECTION: Establishment of Plant Cultures
1.1. Collect dormant shoots, cut them (3 cm in length),and cleanse them by submerging in 70% ethanol for 1 minute, followed by rinsing with sterile deionized water. Then, immerse the shoots in a 10% bleach solution for 10 minutes, and rinse them twice with deionized water (Figure 1).

 

SECTION: Establishment of Plant Cultures
1.2. Peel the vegetative shoot buds and transfer them into culture tubes containing 20 mL of Murashige and Skoog agar media. Place the shoot vertically ensuring that the bud is 1 cm above the agar media.
SECTION: Establishment of Plant Cultures
2. Establishment of plant cultures from seeds
SECTION: Establishment of Plant Cultures
2.1. Clean fruit exocarps with 20% bleach for 10 minutes, followed by a 10-minute immersion in 70% ethanol.
SECTION: Establishment of Plant Cultures
3. Maintenance of stock plants
SECTION: Establishment of Plant Cultures
2.2. Extract seeds within a laminar flow hood and transfer them aseptically into culture tubes containing Woody Plant Medium. Allow them to undergo stratification for ten weeks in darkness at 4 ºC.
SECTION: Establishment of Plant Cultures
2.3. Upon germination (Figure 2), micropropagate shoot tips in culture vessels(Magenta GA-7) using an agar-based medium

 

SECTION: Establishment of Plant Cultures
3.1. Sustain stock plants in Magenta GA-7 vessels by transferring shoot tips every five weeks onto a fresh medium.
SECTION: Establishment of Plant Cultures
3.2. Maintain vessels under a photosynthetic photon flux density of 20 μmol/s/m2, with a 16-hour photoperiod at 24°C.

Optional: When the When the presence of hyper multiplication, an occasional resting cycle with 16 μM indole-3-acetic acid (resting media) is recommended.
SECTION: Establishment of Plant Cultures
3.3. 
SECTION: Fungi Preservation
4. Propagate fungal cultures in Petri plates by placing two plugs (0.5 × 0.5 cm) from the youngest part of the colony (Figure 3).

 

SECTION: Fungi Preservation
5. Seal plates with parafilm and maintain in the dark at 20 ºC.
SECTION: Fungi Preservation
6. Refresh every 14 days by transferring mycelial plugs to fresh MEA plates to ensure active fungal growth.
SECTION: Inoculum preparation
7. Extract three ten-millimeter-diameter plugs from the edge of two-week-old colonies
SECTION: Inoculum preparation
8. Remove most of the agar plug and homogenize mycelium with 5 mL of sterile water using a sterilized 15 mL tissue grinder (Figure 4).

 

SECTION: Inoculum preparation
9. Place a sterile ultra-clear porous cellophane sheet on top of a Petri dish containing malt extract agar media and pour 600 µL of homogenate
SECTION: Inoculum preparation
10. Spread homogenate uniformly over the entire plate using a cell spreader and incubate the plate in the dark for 14 days.
SECTION: Inoculum preparation
11. Prepare the mycelium suspension for inoculum by taking a 2 × 2 cm plug from the previously prepared
plate and homogenate with 7 mL of sterile water using a tissue grinder, and aliquoted in 1.5 mL Eppendorf tubes (Figure 5).

 

SECTION: Co-culture (plant-fungi) establishment
12. Autoclave (121 ºC for 20 min) rectangular plastic vessels (110 × 297 mm; Southern Sun
BioSystems) and after cooling down to room temperature, set the fiber-supported
paper on the floor of each vessel (Figure 6).

 

 

SECTION: Co-culture (plant-fungi) establishment
13. Add 175 mL of plant growth regulator-free liquid ‘New Prunus Medium’ to each vessel.
SECTION: Co-culture (plant-fungi) establishment
14. Transfer fifteen in vitro plants from an agar-based medium to each vessel removing the agar and sealing vessels with polyvinyl chloride film (Figure 7).

 

SECTION: Co-culture (plant-fungi) establishment
15. Place vessels on a rocker's arm with an articulated shelf that providesone swing every 15 min.
SECTION: Co-culture (plant-fungi) establishment
16. Use another set of rectangular Southern Sun BioSystems vessels containing 175 mL of ‘New Prunus Medium’, fiber matte, and germination paper and add 1 mL of the mycelium suspension for inoculum.
SECTION: Co-culture (plant-fungi) establishment
17. Seal the vessels with PVC film and place them on an automatic rocker arm at 5 rpm under μM/m2/s LED light 2 red 1 blue and 16 h/day photoperiod at 24 ºC.
SECTION: Co-culture (plant-fungi) establishment
18. After ten and seventeen days of inoculation with A. mellea and D. caespitosa, respectively, transfer the in vitro rooted plants from the liquid media to the inoculated vessels.
SECTION: Co-culture (plant-fungi) establishment
19. Add 60 mL of ‘New Prunus Medium’ without any plant growth regulator just before plant transferring.
SECTION: Co-culture (plant-fungi) establishment
20. Seal the vessels with PVC film and place them on an automatic rocker arm at 5 rpm under μM/m2/s LED light 2 red 1 blue and 16 h/day photoperiod at 24 ºC (Figure 8).

 

SECTION: Co-culture (plant-fungi) establishment
21. Collect tissues when needed.