Jul 03, 2024

Public workspaceIn vitro co-culture system using a fiber-supported liquid approach

DOI
dx.doi.org/10.17504/protocols.io.rm7vzj56xlx1/v1
  • Alejandro Calle1,
  • Jeffrey Adelberg2,
  • Guido Schnabel2,
  • Jacqueline Naylor-Adelberg2,
  • Jhulia Gelain2,
  • Yeter Karakoc2,
  • Jared Weaver2,
  • Christopher Saski2,
  • Ksenija Gasic2
  • 1Fruit Production Program, Institut de Recerca i Tecnologia Agroalimentàries (IRTA);
  • 2Department of Plant and Environmental Sciences. Clemson University. Clemson, SC, USA
Ksenija Gasic
Open access
DOI: dx.doi.org/10.17504/protocols.io.rm7vzj56xlx1/v1
Protocol Citation: Alejandro Calle, Jeffrey Adelberg, Guido Schnabel, Jacqueline Naylor-Adelberg, Jhulia Gelain, Yeter Karakoc, Jared Weaver, Christopher Saski, Ksenija Gasic 2024. In vitro co-culture system using a fiber-supported liquid approach. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzj56xlx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 02, 2024
Last Modified: July 03, 2024
Protocol Integer ID: 102750
Keywords: Phenotyping, plant disease infection, fungal inoculation, plant culture
Funders Acknowledgement:
USDA-NIFA
Grant ID: 2020-51181-32142.
Abstract
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.
Materials
Materials
  • 20 × 150 mm culture tubes (Stellar Scientific; Baltimore, MD, USA, Cat. Nº: SKU:GS-1522)
  • Autoclavable polypropylene culture tube closures (General Laboratory Supply; Pasadena, TX, USA, Cat. Nº: T3054-4)
  • Magenta‱ GA-7 vessels (Merck, Darmstadt, Germany, Cat. Nº: V8505-25EA)
  • Petri plates (VWR International, Radnor, PA, USA, Cat. Nº: 391-0579)
  • Parafilm ‘M’ laboratory film (Sigma Aldrich; Darmstadt, Germany; Cat. Nº: P7668)
  • 15 mL Pyrex‱ Ten Broeck tissue grinder with a pour spout (Corning, Tewksbury, MA, USA; Cat. Nº: 7727-15)
  • Ultra-clear porous cellophane sheet (0.1 mm thick) (Research Products International, Mount Prospect, IL, USA; Cat. Nº: 1080)
  • Lazy-L spreader (Merck, Darmstadt, Germany, Cat. Nº: Z376779)
  • 1.5 mL Eppendorf‱ tubes (Thermo Fisher Scientific Inc, Waltham, WA, USA; Cat. Nº: 0030120175)
  • Rectangular vessels (110 × 297 mm; Southern Sun BioSystems, Hodges, SC, USA)
  • Polyester fiber matte (BioStrate‱ Felt; Cropking Inc., Lodi, OH, USA)
  • Germination paper (Anchor Paper Co., St. Paul, MN, USA)
  • Polyvinyl chloride (PVC) sealing film (Phytotech Laboratories, Shawnee Mission, KS, USA; Cat. Nº: A003)

Equipment
  • Laminar flow hood
  • Autoclave
  • Articulated rocker arm
  • Fungal growth incubator
  • LED light NutriLED, Hubbell Lighting, Greenville, SC, USA)
Establishment of Plant Cultures
Establishment of Plant Cultures
Establishment of plant cultures from dormant shoots
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).

Figure 1. Cleaning process of dormant shoots
Figure 1. Cleaning process of dormant shoots

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.
Establishment of plant cultures from seeds
Clean fruit exocarps with 20% bleach for 10 minutes, followed by a 10-minute immersion in 70% ethanol.
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.
Upon germination (Figure 2), micropropagate shoot tips in culture vessels(Magenta GA-7) using an agar-based medium

Figure 2. Germinated seeds in tubes.
Figure 2. Germinated seeds in tubes.

Maintenance of stock plants
Sustain stock plants in Magenta GA-7 vessels by transferring shoot tips every five weeks onto a fresh medium.
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.

Fungi Preservation
Fungi Preservation
Propagate fungal cultures in Petri plates by placing two plugs (0.5 × 0.5 cm) from the youngest part of the colony (Figure 3).

Figure 3. One-week-old Armillaria mellea cultures
Figure 3. One-week-old Armillaria mellea cultures

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

Figure 4. Fungi homogenization in sterile water
Figure 4. Fungi homogenization in sterile water

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
Spread homogenate uniformly over the entire plate using a cell spreader and incubate the plate in the dark for 14 days.
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).

Figure 5. Preparation of the inoculum suspension for infection.
Figure 5. Preparation of the inoculum suspension for infection.

Co-culture (plant-fungi) establishment
Co-culture (plant-fungi) establishment
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).

Figure 6. a) Fiber-supported paper.
Figure 6. a) Fiber-supported paper.

Figure 6. b) Southern Sun BioSystems with fiber-supported paper inside.
Figure 6. b) Southern Sun BioSystems with fiber-supported paper inside.

Add 175 mL of plant growth regulator-free liquid ‘New Prunus Medium’ to each vessel.
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).

Figure 7. In vitro plants growing in the Southern Sun BioSystems vessels.
Figure 7. In vitro plants growing in the Southern Sun BioSystems vessels.

Place vessels on a rocker's arm with an articulated shelf that providesone swing every 15 min.
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.
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.
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.
Add 60 mL of ‘New Prunus Medium’ without any plant growth regulator just before plant transferring.
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).

Figure 8. Southern Sun BioSystems vessels on rocking platform with an articulated shelf (5rpm) exposed to 20 µmol/s/m², 16h/day at 24 ºC.
Figure 8. Southern Sun BioSystems vessels on rocking platform with an articulated shelf (5rpm) exposed to 20 µmol/s/m², 16h/day at 24 ºC.

Collect tissues when needed.
Protocol references
Adelberg J, Simpson EP. Intermittent immersion vessel apparatus and process for plant propagation. US Patent 2004; 6: 753, 178
Adelberg J, Naylor-Adelberg J, Miller S, Gasic J, Schnabel G, Bryson P, et al. In vitro co-culture system for Prunus spp. and Armillaria mellea in phenolic foam rooting matric. Plant 2021; 57: 387-397. https://doi.org/10.1007/s11627-020-10136-2
Adelberg J, Naylor-Adelberg J, Rapaka V. A novel rooting matrix and vessel system resulted in larger plants and faster growth during greenhouse acclimatation of Hydrangea quercifolia ‘Sikes dwarf’. Propagation of Ornamental Plants 2015; 15(3): 89-94.
Baumgartner K. Root collar excavation for postinfection control of Armillaria root disease of grapevine. Plant Dis. 2004; 88: 1235–1240. https://doi.org/10.1094/PDIS.2004.88.11.1235
Baumgartner K, Fujiyoshi P, Ledbetter C, Duncan R, Kluepfel D. Screening almond rootstocks for sources of resistance to Armillaria root disease. HortScience 2018; 53: 4–8. https://doi.org/10.21273/HORTSCI12038-17
Beckman TG, Okie WR, Nyczepir AP, Pusey PL, Reilly CC. Relative susceptibility of peach and plum germplasm to Armillaria root rot. HortScience 1998; 33: 1062–1065. https://doi.org/10.21273/HORTSCI.33.6.1062
Beckman TG, Pusey PL. Field testing peach rootstocks for resistance to Armillaria root rot. HortScience 2001; 36: 101–103. https://doi.org/10.21273/HORTSCI.36.1.101
Beckman TG, Chaparro JX, Sherman WB. ‘MP-29’, a clonal interspecific hybrid rootstock for peach. HortScience 2012; 47:128–131. https://doi.org/10.21273/HORTSCI.47.1.128
Cai L, Adelberg J, Naylor-Adelberg J, Schnabel G, Calle A, Li Z, et al. Transcriptomic reveal the genetic coordination of early defense to Armillaria root rot (ARR) in Prunus spp. Front. in Plant Sci. 2023; 14: 1181153.
Devkota P, Hammerschmidt R. A rapid and holistic approach to screen susceptibility of Prunus species to Armillaria root rot. Forest Pathol 2019; 49. https://doi.org/10.1111/efp.12547
Devkota P, Hammerschmidt R. The infection process of Armillaria mellea and Armillaria solidides. Phys. and Mol. Plant Path. 2020; 112: 101543. https://doi.org/10.1016/j.pmpp.2020.101543
Devkota P, Iezzoni A, Gasic K, Reighard G, Hammerschmidt R. Evaluation of susceptibility of Prunus rootstock genotypes to Armillaria and Desarmillaria species. Eur. J. Plant Pathol. 2020; 158: 177-193. https://doi.org/10.1007/s10658-020-02065-y
Economou AS. From microcutting to microplant establishment: key points to consider for maximum success in woody plants. Acta Hort 2013; 988: 43–56. https://doi.org/10.17660/ActaHortic.2013.988.3
Fenning TM. The use of tissue culture and in vitro approaches for the study of tree diseases. Plant Cell, Tissue and Organ Culture 2019; 136: 415-430. https://doi.org/10.1007/s11240-018-01531-0
Gasic K, Adelberg J, Baumgartner K, Brannen PM, Cai L, Calle A, et al. Solutions to the Armillaria root rot affecting the US stone fruit industry. Acta Hortic. 2022; 1352, 501-508 https://doi.org/10.17660/ActaHortic.2022.1352.68
Guillaumin JJ, Pierson J, Grassely C. The susceptibility of different Prunus species used as stone fruit rootstocks to Armillaria mellea (sensu stricto). Seventh International Conference on Root and Butt Rots. International Union of Forestry Research Organizations, Vernon and Victoria, BC, Canada 1989.
Hao J, Yang ME, Davis RM. Effect of soil inoculum density of Fusarium oxysporum f. sp. vasinfectum Race 4 on disease development in cotton. Plant Dis. 2009; 93(12) 1324-1328. https://doi.org/10.1094/PDIS-93-12-1324
Herrin G. An economic impact and investment analysis of Armillaria root rot in the United States peach industry. All Theses 2022; 3949. https://tigerprints.clemson.edu/all_theses/3949
Hood IA, Redfern DB, Kile GA. Armillaria in planted hosts. In Armillaria root disease, USDA Forest Service, pp. 1991; 122-149.
Lebeda A, Švábová L. In vitro screening methods for assessing plant disease resistance. In Mass screening techniques for selecting crops resistant to disease. FAO/IAEA. Vienna, 2010. Pp 5-45.
Lloyd G, McCown B (1981) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Comb Proc Intl Plant Prop Soc 2010; 30: 421-427.
Mansilla JP, Aguin O, Sainz MJ. A fast method for production of Armillaria inoculum. Mycologia 2001; 93: 612–615. https://doi.org/10.1080/00275514.2001.12063191
Miller SB, Gasic K, Reighard G, Henderson WG, Rollins PA, Vassalos, M. et al. Preventative root-collar excavation reduces peach tree mortality caused by Armillaria root rot on replant sites. Plant Dis. 2020; 104(5): 1274–1279. https://doi.org/10.1094/PDIS-09-19-1831-RE
Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 1962; 15(3): 473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Parris SM, Jeffers SN, Olvey JM, Adelberg JW, Wen L, Udall JA, et al. An in vitro co-culture system for rapid differential response to Fusarion oxysporum f. sp vasinfectum Race 4 in three cotton cultivars. Plant Dis 2022; 106(3):990-995. https://doi.org/10.1094/PDIS-08-21-1743-RE
Pérez-Clemente RM, Gómez-Cadenas A. In vitro tissue culture, a tool for the study and breeding of plants subjected to abiotic stress conditions. In Recent Advances in Plant in vitro Culture 2012; 91-108. https:// doi.org/10.5772/50671
Raabe RD. Testing grape rootstocks for resistance to the oak root fungus. California Plant Pathol 1979; 46: 3-4.
Raziq F, Fox RTV. Combinations of fungal antagonists for biological control of Armillaria root rot of strawberry plants. Biol Agr Hort 2005; 23: 45–57. https://doi.org/10.1080/01448765.2005.9755307
Schnabel G, Agudelo P, Henderson GW, Rollins PA. Aboveground root collar excavation of peach trees for Armillaria root rot management. Plant Dis. 2012; 96: 681–686. https://doi.org/10.1094/PDIS-06-11-0493
Schnabel G, Ash JS, Bryson PK. Identification and characterization of Armillaria tabescens from the southeastern United States. Mycol. Res. 2005; 109: 1208–1222. https://doi.org/10.1017/s0953756205003916
Shishido K, Murakami H, Kanda D, Fuji S, Toda T, Furuya H. Effect of soil inoculum density and temperature on the incidence of Cucumber black root rot. Plant Dis. 2015; 100(1): 5-235 https://doi.org/10.1094/PDIS-12-14-1287-RE
Tascan A, Adelberg JW, Tascan M, Rimando A, Joshee N, Yadav A. Hyperhydricity and flavonoid content of Scutellaria species in vitro on polyester-supported liquid culture systems. HortScience 2010; 45(11): 1723-1728. https://doir.org/10.21273/HORTSCI.45.11.1723
Tascan A, Adelberg JW, Joshee N, Yadav AK, Tascan M. Liquid culture system for Scutellaria species. Act Hort 2007; 756: 163-170. https://doi.org/10.17660/ActaHortic.2007.756.18
Thomas HE, Roberts C, Amstutz A. Rootstock susceptibility to Armillaria mellea. Phytopathology 1948; 38:152–154.
Zewdu G, Tamene W, Assefa Z, Tamiru T, Bankseni L, Borja M, et al. The role of modern plant breeding to control plant disease: a review. Agricultural J. 2022; 17(4): 19-27. https://doi.org/10.1016/j.tifs.2020.03.042