An Optimised Greenhouse Protocol for Screening Banana Plants for Fusarium Wilt Resistance

Abstract

Fusarium wilt, caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc), is considered one of the most devastating diseases of banana in the world. Effective management of Fusarium wilt is only achieved by planting banana varieties resistant to Foc. Resistant bananas, however, require many years of breeding and field-testing under multiple geographical conditions. Field evaluation is reliable but time consuming and expensive. Small plant screening methods are, therefore, needed to speed up the evaluation of banana varieties for Foc resistance. To this end, a small plant screening method for resistance to banana Fusarium wilt is presented. The method proposes the planting of 2- to 3-month-old banana plants in soil amended with 10 g Foc-colonised millet seeds. Rhizome discoloration is then evaluated to rank the disease resistance response. The optimized millet seed technique could be useful in mass screening of newly developed genotypes for resistance to Foc.

1 Introduction

Fusarium wilt is considered one of the most devastating diseases of banana in the world (Stover 1962; Ploetz 2006). The disease is caused by a soil-borne fungus, Fusarium oxysporum f. sp. cubense (Foc), which infects the plants through the roots, colonises the rhizome and xylem, and causes a lethal wilting of plants (Stover 1962; Guo et al. 2015; Warman and Aitken 2018). Foc originated in Southeast Asia, and was disseminated globally with Foc-propagating and planting material as banana production expanded during the twentieth century (Stover 1962; Ploetz 2015a; Dita et al. 2018). By the 1950s, Fusarium wilt was so widespread in Latin America that it became impossible to sustain the international banana export industry that was almost exclusively based on the Gros Michel (AAA) bananas. Gros Michel was thus replaced with Cavendish bananas as export variety in the 1960s, which currently account for nearly 50% of production globally (Lescot 2015; Ploetz 2015a). Cavendish bananas, however, are now also severely affected by a different strain of Foc in Asia, the Middle East, Mozambique in Africa and Colombia in Latin America, called Foc Tropical Race 4 (TR4) (Ploetz and Pegg 2000; Mostert et al. 2017; Dita et al. 2018; García-Bastidas et al. 2019a, b; Thangavelu et al. 2019; Viljoen et al. 2020).

Foc comprises of three races based on their pathogenicity to a group of differential cultivars, with Foc races 1, 2 and 4 causing disease to Gros Michel, Bluggoe and Cavendish bananas, respectively (Pegg and Langdon 1987; Ploetz and Pegg 2000). Foc race 4 is further subdivided into Foc subtropical race 4 (Foc STR4) and tropical race 4 (Foc TR4) strains. The former causes disease on Cavendish bananas in the subtropics when plants are stressed by adverse climatic conditions, while Foc TR4 affects Cavendish bananas both under tropical and subtropical conditions (Ploetz and Pegg 2000). Foc TR4 has a wider host range than Foc races 1 and 2, and causes disease to Cavendish banana cultivars as well as most Foc race 1- and race 2-susceptible cultivars (Ploetz 2015b). Foc strains have been further classified using vegetative compatibility group (VCG) analysis, which is based on the ability of fungal hyphae to anastomose and form a stable heterokaryon (Leslie and Summerell 2006). Twenty-four VCGs have been described for Foc (Pegg et al. 1993; Bentley et al. 1995; Ploetz and Correll 1998; Fourie et al. 2009).

The most effective method to control banana Fusarium wilt is the use of Foc-resistant varieties (Ploetz 2015b). If existing varieties with resistance are not available to replace susceptible ones, the susceptible bananas can be improved by conventional breeding, mutation breeding, genetic engineering and genome editing (Rowe 1987; Hwang and Tang 2000; Hwang and Ko 2004; Bakry et al. 2009; Ortiz 2013; Dale et al. 2017; Naim et al. 2018). Bananas resistant to Foc race 1 that have been developed by conventional breeding include the hybrids FHIA-01, FHIA-18 and FHIA-25 developed in Honduras; RG1, Bodles Altafort, 2390-2 and 72-1242 bred in Jamaica; Pacovan Ken, Preciosa and Tropical in Brazil; and BRS-01 and BRS-02 in India (Bakry et al. 2009; Lorenzen et al. 2012; Ortiz 2013). Several partially resistant and/or resistant clones have also been developed by mutation breeding through chemical and radiation mutagenesis techniques and through somaclonal variation. For instance, GCTCV-215, GCTCV-105, GCTCV-218 and GCTCV-219 are Giant-Cavendish derived somaclones, which were selected through somaclonal variation, and Dwarf Parfitt mutant DPM-25 is a banana mutant generated with gamma irradiation (Tang and Hwang 1994; Hwang and Ko 2004; Smith et al. 2006). Dale et al. (2017) developed two transgenic Cavendish lines, RGA2-3 and Ced9-21, which did not develop any Fusarium wilt after 3 years of evaluation in banana fields infested with Foc TR4. Apart from GCTCV-218, most improved bananas with Fusarium wilt resistance have, however, not satisfied export market requirements (Bakry et al. 2009; Ortiz 2013).

Before local varieties or improved bananas can replace susceptible bananas in Foc-infested fields, they need to be evaluated for resistance against local Foc strains (Morpurgo et al. 1994a; Ploetz 1994; Carlier and De Waele 2002; Mak et al. 2004; Dita et al. 2011). Field evaluation is more accurate when identifying Foc-resistant plants under natural environmental conditions, but the process is time-consuming and expensive. Inoculum levels in the soil might also be unequally distributed. In addition, banana varieties can only be tested against Foc strains present in the country or region where the tests are performed. Greenhouse screening can be performed on small plants, achieved in a short time, and can be screened against quarantine pathogens in restricted environments (Smith et al. 2008; Dita et al. 2011). Yet, greenhouse screening methods seldom correlate well with field screening due to a number of factors, including inoculum preparation, inoculum concentration, inoculation method, the effect of temperature and photoperiod, type of planting material tested, and the potting soil used (Brake et al. 1995; Smith et al. 2008; Dita et al. 2011). Greenhouse screening methods, thus, need optimisation.

Several greenhouse evaluation methods have been developed for banana Fusarium wilt (Morpurgo et al. 1994b; Brake et al. 1995; De Ascensao and Dubery 2000; Mohamed et al. 2001; Smith et al. 2008; Wu et al. 2010; Dita et al. 2011; Viljoen et al. 2018; Chen et al. 2019; García-Bastidas et al. 2019a, b). The most common inoculation methods include the dipping of plant roots in a conidial suspension, the drenching of soil with a conidial suspension, and the replanting of plants in Foc-infested soil or sand. In the dipping method, plants are carefully removed from soil and their roots dipped in a conidial suspension for a few minutes before replanting (Mohamed et al. 2001; Dita et al. 2011; Ribeiro et al. 2011). The soil-drenching method consists of pouring a spore suspension on the surface of potting soil (Smith et al. 2008). In the infested soil technique, bananas are planted in soil mixed with millet seeds or maize kernels that were pre-colonized with Foc (Smith et al. 2008; Dita et al. 2011). A combination of the dipping method and replanting in infested soil was reported to result in quick and consistent disease development (Dita et al. 2011). The screening of banana plants grown in vitro has also been reported, but Hamill (2018) considered these not suitable for resistance screening, as the fungus may kill both susceptible and resistant varieties due to high inoculum pressure.

The type of planting material used can influence Fusarium wilt development in the greenhouse. Tissue culture-derived plants are more susceptible and have a shorter incubation period compared to suckers and bits (Hwang and Ko 1987; Smith et al. 2008). Tissue culture plants are also free of other pests and diseases, apart from plant viruses, which may influence disease development. Tests with tissue culture plants smaller than 5 cm did not reflect field results, but plants of 10–15 cm (around 2 months old) did (Brake et al. 1995; Mohamed et al. 2001; Smith et al. 2008). Further investigations of the effect of plant age on Fusarium wilt development are thus needed to improve the reliability of small plantlet screening methods (Brake et al. 1995; Smith et al. 2008; Ribeiro et al. 2011).

In this chapter, we present an optimised greenhouse screening method for resistance to banana Fusarium wilt, considering the effect of inoculum concentration, inoculation methods and plant age on disease development.

2 Materials

2.1 Preparation of Plant Material

• 1. Potting bags (3–5-l capacity).

• 2. Commercial potting soil (mix of screened compost, coarse sand and screened bark).

• 3. Building sand.

• 4. Coco peat.

• 5. Slow-release fertiliser.

• 6. Sporekill^® (120 g/L didecyldimethyl-ammonium chloride) solution (ICA International Chemicals (PTY) Ltd., Stellenbosch, South Africa).

• 7. Seedling trays if plants are to be hardened-off.

2.2 Culture Medium

• 1. Potato dextrose agar (PDA) powder.

• 2. Deionised water.

• 3. Petri dishes (90-mm-diameter).

• 4. Analytical balance.

• 5. Weighing trays.

• 6. Spatula.

• 7. 1l Schott bottles.

• 8. Autoclave.

2.3 Preparation of Inoculum and Greenhouse Infection

• 1. Millet seed (Panicum miliaceum or Eleusine corocana).

• 2. 1l Schott bottle or 250 ml Erlenmeyer flasks.

• 3. Sterile distilled water.

• 4. Autoclave.

• 5. Scalpel.

• 6. Culture media (PDA).

• 7. Fusarium oxysporum f. sp. cubense isolate characterised to VCG level. Foc isolates can be obtained from the Westerdijk Institute in the Netherlands (https://wi.knaw.nl/page/Collection) and the Agricultural Research Service of the United States Department of Agriculture (https://nrrl.ncaur.usda.gov/).

• 8. Incubator with light and temperature control (light regime 65 μmol/m²/s; e.g. cool-white fluorescent tubes, Philips TLP 36/86; temperature regime of 25 °C +/− 2 °C).

• 9. Analytical balance.

• 10. Greenhouse (temperature of 25 °C +/− 2 °C and relative humidity of 80%).

2.4 Disease Rating

• 1. Sterile tissue paper.

• 2. Ethanol for surface and tools sterilisation.

• 3. Forceps.

• 4. Scalpels.

• 5. Scalpel blades.

• 6. Knives.

• 7. 15 ml Falcon tubes.

3 Methods

3.1 Planting Material

• 1. Banana accessions to be tested are prepared together with resistant and susceptible control plants. Resistant and susceptible control varieties are selected according to the Foc strain to be used for the inoculation (Table 5.1) (Viljoen et al. 2017).

• 2. Nine to 15 plants per accession are needed for resistance screening. Additional susceptible control plants need to be included in the experiment to monitor the development of internal symptoms.

• 3. Plants must be produced in tissue culture, and the rooted plants weaned for 4–6 weeks in a humidity chamber (see Fig. 5.1). Light conditions are reduced to 20–30% transmission, and humidity increased to 60–90%.

• 4. Roots of the tissue cultures plants are gently washed with a Sporekill solution (1 ml Sporekill/l water) to remove all adhering media and to prevent opportunistic infections.

• 5. Excess leaf and root material is trimmed back, leaving only two to four soft green leaves and several white primary roots (see Fig. 5.2a). The plants are then replanted in plastic seedling trays filled with semi-sterile coco peat amended with a slow-release fertilizer (Osmocote start, 11:11:17 + 2 MgO + TE, 2 g/kg coco peat). The misting system is set to run for 15–20 s every 30 min in the warmer months, and 10 s every 30 min during cooler months.

• 6. Once the weaned plants have reached a height of 5–12 cm (see Fig. 5.2b), they are replanted into planting bags or pots containing a semi-sterile potting mix (commercial soil:sand:peat at 1:1:1) amended with slow release fertiliser (Osmocote Pro, 19:9:10 + 2MgO + TE, 2 g/kg soil mix) to support growth and development. The plants are kept on tables or raised structures in the greenhouse, and irrigated twice daily with an overhead micro-sprinkler system.

• 7. Plants in the hardening-off phase are ready to be screened after a period of 6–12 weeks, or once the plants have reached a height of 25–30 cm (see Fig. 5.2c).

Table 5.1 Banana varieties used as resistant and susceptible checks for evaluation against races of Fusarium oxysporum f. sp. cubense

Fig. 5.1

Construction of a humidity chamber used for the weaning of tissue culture plants. The humidity chamber is constructed out of a metal framework covered in clear polyethylene sheeting. The humidity chamber is covered with shade netting and fitted with an automatic misting system. Seedling trays are placed on a lifted metal mesh to ensure the drainage of excess water

Fig. 5.2

Plants during the weaning and hardening phases. (a) Tissue culture plant ready for weaning. (b) A plant at the end of the weaning phase that is ready to be hardened-off. (c) Hardened-off plants ready to be screened for Fusarium wilt resistance

3.2 Preparation of Culture Media

• 1. Add 39 g of PDA powder to 1l Schott bottle and fill up with deionised water.

• 2. Autoclave at 121 °C for 20 min.

• 3. Cool down to 50 °C.

• 4. Poor into 90-mm sterile Petri dishes.

• 5. Store PDA plates at 4 °C until use.

3.3 Millet Seed Inoculum Preparation

• 1. Fill 1l Erlenmeyer flasks or Schott bottles with 250 g millet seeds.

• 2. Add 200 ml of distilled water to soak the millet seeds overnight.

• 3. Drain-off all excess water from the millet seeds the following morning.

• 4. Autoclave the millet seed at 121 °C for 20 min on two consecutive days.

• 5. Shake the flask/bottle to loosen the grain (see Fig. 5.3a). A small sample is collected for plating onto culture media to ensure that it is sterile.

• 6. Plate out a Foc isolate on PDA and incubate it at 25 °C for 5–7 days.

• 7. Cut 10 mycelial plugs of 0.5 cm in diameter from the margins of the culture, and transfer these to the flasks/Schott bottles containing sterilized millet seeds.

• 8. When white fungal growth begins to show on the surface of millet seeds, the flasks/bottles are shaken to distribute the fungus and to prevent the kernels from clumping together (see Fig. 5.3b).

• 9. Let the fungus colonise the millet seed for 14 days, but shake every second day to ensure thorough colonisation of the millet seed. Plate out some seeds onto PDA to ensure that it is colonised by Foc only (see Fig. 5.3c). The millet seeds can then be stored at room temperature until use, but not for longer than 3 months to ensure the fitness and pathogenicity of Foc.

Fig. 5.3

Preparation of Fusarium oxysporum f. sp. cubense (Foc) inoculum on millet seed. (a) Autoclaved millet seeds ready for inoculation, (b) white fungal growth on the surface of the millet seeds. At this time, the flasks/Schott bottles need to be shaken to ensure proper distribution and thorough colonisation of millet seeds, and (c) the plating out of Foc-colonized millet seeds onto PDA plates to ensure proper colonization with the Foc inoculum, and no contamination with other micro-organisms

3.4 Greenhouse Inoculation

• 1. Fill planting bags/pots halfway with steam-sterilized potting soil, and mix with the Foc inoculum at a rate of 10 g millet seeds/kg soil (see Fig. 5.4a, b).

• 2. Uproot the plant (2-month-old) that needs to be inoculated carefully, and replant it into potting bags/pots containing the Foc-inoculated potting soil (see Fig. 5.4c). Then fill up the entire bag/pot with potting soil. Clearly mark the bags/pots with the accession name.

• 3. Once all plants are inoculated, they need to be randomly arranged in the greenhouse (see Fig. 5.4d).

• 4. Experimental conditions should include a 12 h daylight photoperiod at 25/20 °C, and 80% humidity.

• 5. Add 1 g of slow-release fertilizer per pot for the screening period, and irrigate plants to prevent any environmental stresses.

Fig. 5.4

Inoculation of banana plants for resistance screening against Fusarium oxysporum f. sp. cubense (Foc). (a) Weighing-off of millet seed inoculum, (b) mixing Foc-millet seeds with potting soil, (c) uprooted plantlet ready for replanting in bags/pots containing Foc-infested soil, (d) inoculated plants set up in a screen house and (e) plants ready for disease development rating

3.5 Scoring Disease Severity

• 1. Assess the host plant response when leaf symptoms are visible on 50% of the susceptible control plants, or when susceptible control plants have an internal disease rating of 5 and more. This is usually 6–12 weeks after inoculation.

• 2. For internal disease rating, the rhizome should be cut open horizontally in the middle of the rhizome, where discoloration is more pronounced than in the lower or upper rhizome (see Fig. 5.5a, b).

• 3. Score the rhizome discoloration on a rating scale ranging from 1 to 6, with 1 indicating no disease symptoms and 6 indicating complete discolored of the inner rhizome (see Fig. 5.5c).

Fig. 5.5

Evaluation of Fusarium wilt development. (a) Rhizomes of inoculated plants are cut open for rating, (b) the position where the cut is made, and (c) rating is based on the discoloration of the inner rhizome as follows: 1 = no internal symptoms, 2 = few internal spots, 3 = <1/3 of the inner rhizome affected, 4 = 1/3–2/3 of the inner rhizome discolored 5 = >2/3 of the inner rhizome discolored and 6 = entire inner rhizome discolored