Keywords

1 Introduction

Banana and plantains belong to the genus Musa and are important agricultural products in developing countries. More than 1000 varieties of bananas are produced and consumed locally. Cavendish banana (AAA) are the main commercial variety for export and international trade and account for around 47% of global production (FAO 2019a). Approximately 50 million tons of Cavendish bananas are being produced globally every year. In 2017 the global banana production reached 114 million tons (FAO 2019b). Bananas are locally consumed as vital staple food or as a significant addition to the diets in Africa, southern Asia, and tropical America (FAO 2019b).

Fusarium oxysporum is a soil-borne fungus that is ranked fifth on the list of top fungal plant pathogens (Ploetz 2005; Dean et al. 2012). Over 120 formae speciales (ff. spp.) of Fusarium oxysporum have been described based on host specificity (Baayen et al. 2000). Differences in pathogenicity on specific host cultivars is being defined as physiological races among isolates (Kistler 1997; Baayen et al. 2000; Takken and Rep 2010; Meldrum et al. 2012). Fusarium oxysporum f. sp. cubense (Foc) refers to strains that infect bananas and plantains and cause Fusarium wilt or Panama disease (Ploetz 2005). It has been recognized that Foc has a polyphyletic origin (Lievens et al. 2009), hence comprises a suite of genetically distinct lineages (Ordonez et al. 2015). Maryani et al. (2019) have recently revised the taxonomy of Foc and designated different species names to strains affecting banana and merged them into the Fusarium of Banana Species Complex.

The disease cycle of this Fusarium spp. starts with infection of the root system and subsequent colonization within the vascular tissue, leads to water stress, severe chlorosis, and wilting (Ploetz 2015). Infected plants frequently die before they produce bunches, hence Fusarium wilt significantly reduces yields in infested fields (Dita et al. 2010).

A variant of Foc, called tropical race 4 (TR4) was first identified in Taiwan in 1989 but was probably the cause of banana wilt in the country since 1960. In the 1990s, Foc TR4 was identified in Malaysia and Indonesia, and the strains are thought to have originated from Taiwan (Buddenhagen 2009; Maryani et al. 2019). Foc TR4 has spread to many countries of Asia, as well as Australia and Africa and recently has reached Colombia and Peru in Latin America. Since its appearance, TR4 has severely affected Cavendish plantations in Malaysia, Indonesia, South China, Philippines, and the Northern Territory of Australia (Ploetz 2006; Molina et al. 2010; Buddenhagen 2009; Chittarath et al. 2018). TR4 is considered one of the most destructive Foc strains because of its broad host range. This pathogen is attacking the important cultivars of Cavendish but also all other cultivars that are sensitive to Foc (Cheng et al. 2019). The disease predominantly affects the Cavendish varieties, which not only primarily meets the international market demand but also is important for local consumption in developing countries. Cavendish varieties are the cornerstone for international trade, therefore TR4 threatens the entire global production (FAO 2019b).

Strategies controlling TR4 spread are based on visual monitoring of early symptom appearance, eradication of infected plants and isolation of infested areas to reduce pathogen dissemination. Pérez Vicente et al. (2014) reported that once plants are infected with TR4, there is no way to eradicate the disease. In this case the affected plants and all plants in the surrounding 7.5 m radius should be destroyed. Host resistance is a basis for sustainable disease management in most crops and this is usually achieved by intensive breeding programs (Ploetz 2006). Therefore, breeding for resistant/tolerant banana plants is the best way to overcome the disease.

To develop new, resistant cultivars, breeders need reliable and rapid phenotyping methods enabling selection of improved lines (García-Bastidas et al. 2019). Different approaches can be pursued for resistance screening e.g. in the field or under greenhouse conditions (Dhingra and Sinclair 1986; Trigiano et al. 2004; Singh and Singh 2005). Field screening encounters problems such as time, costs, variable environmental conditions, and unspecified biodiversity of soil-borne pathogens (Mert and Karakaya 2003; Subramaniam et al. 2006; Sutanto et al. 2013). In contrast, greenhouse-based phenotyping facilitates high-throughput selection under controlled conditions with specific fungal genotypes, leading to more reproducible results (Smith et al. 2008). Greenhouse assessments have been reported as a reliable method by several researchers (Smith et al. 2008; Pérez Vicente et al. 2014). In vitro screening is one of the most high-throughput and efficient method (Švábová and Lebeda 2005; Pillay 2002; Naserian Khiabani et al. 2018; Wu et al. 2010). Compared to selection in an experimental field, in vitro selection can considerably reduce the space needed for screening. However, some factors influencing in vitro selection may differ from those in field selection (Matsumoto et al. 2010). The most used selection agents in the tissue culture medium are metabolites of pathogens or similar chemicals. There have been several reports of the use of fusaric acid to select Fusarium resistance in in vitro culture system (Matsumoto et al. 2010; Wu et al. 2010; Švábová and Lebeda 2005). Daub (1986) used a crude filtrate of a Fusarium suspension as a selection agent under the in vitro condition. Wu et al. (2010) and Naserian Khiabani et al. (2018) used suspensions containing pathogenic components including micro and macro-conidia and mycelium of Fusarium oxysporum, as a selection agent for in vitro screening. Using methionine sulfoximine as a selective agent, Carlson (1973) demonstrated for the first time the possibility of selecting disease-resistance plants using an in vitro tobacco protoplast system. Since 1980, the theoretical and practical approaches of in vitro selections and their usefulness for plant breeding have been addressed (Shepard 1981; Wenzel 1985; Daub 1986; Buiatti and Ingram 1991; Švábová and Lebeda 2005). According to Lebeda and Svábová (2010) the ideal system for in vitro selection for disease resistance should comprise: (1) an in vitro explant culture that can generate genetic variations (or an in vitro mutation induction system) with efficient recovery of genetically stable and fertile resistant/tolerant plants; (2) a selection agent that can be readily produced and which induces similar biochemical reactions in vitro as the pathogen in vivo; and, (3) molecular tools to characterize the selected resistant lines at the DNA level.

Several successful experiments have been carried out in vitro with live inoculums. For example: Clavibacter michiganensis (Bulk et al. 1991); Xanthomonas campestris (Hammerschlag 1990); Mycosphaerella musicola (Trujillo and Garcia 1996); Alternaria alternata (Takahashi et al. 1992); Fusarium solani (Huang and Hartman 1998); and Phytophthora cinnamoni (McComb et al. 1987; Cahill et al. 1992). Matsumoto et al. (2010) used fusaric acid as a selection agent in an in vitro culture system to select banana plants resistant to Fusarium wilt race 1.

Basic knowledge about the biology of the causal agent and its relationship with the host plant is necessary to develop suitable methods for resistance screening and selection (Russell 1978). Usually, wilting is either caused by blockage of plant vessels due to the accumulation of spores and mycelium of pathogenic fungi or due to a toxic element produced by the pathogen. In case of Fusarium, the use of fusaric acid or a crude filtered fungal extract does not lead to blockage of the xylem vessels. In this case, it is the toxicity of the extract that leads to the appearance of symptoms. This is distinct from disease resistance screening under field conditions, where plants are affected by the live pathogen, spores, mycelia in addition to toxins. In vitro plantlets inoculated with mycelium and spores of the pathogen are expected to show symptoms very similar to those observed in the field.

To accelerate Fusarium wilt resistance screening in banana breeding programs, bioassays that can efficiently and accurately differentiate resistant from susceptible cultivars are required. Two prerequisites should be met for effective in vitro disease resistance screening using a pathogen-derived selection agent: (1) One or more compounds found in the selection agent should be present in infected plants; and (2) the agent should at least partially induce disease symptoms when inoculated into healthy plants. When an in vitro plantlet is directly inoculated by the pathogen (conidia and mycelium), the above-mentioned requirements are met.

Traditional banana breeding is faced with several impediments, primarily the sterile nature of the triploid cultivars; seedless fruits are required to meet consumer demands but hampers breeding (Pillay and Tenkouano 2011; Pillay 2002). Banana breeders have incorporated non-classical breeding approaches, such as mutation breeding, to induce diversity in their elite germplasm. Mutagenic agents, such as radiation or certain chemicals, can be used to generate genetic variation from which desired mutants may be selected. The combination of mutation breeding and in vitro culture (also called in vitro mutagenesis) is effective for the induction and selection of somatic mutations (Roux 2004). Novák et al. (1989) described the dose-response of tissue-cultured shoot tips to gamma irradiation. Since edible bananas are vegetatively propagated and heterozygous, mutation breeding is an ideal approach for their genetic improvement (Jain 2010). In addition, mutagenic treatments need to be optimized and efficient screening techniques developed to select desirable mutants (Jain 2000, 2006, 2007; Van Harten 1998). In vitro techniques can improve the effectiveness of mutation induction, especially when handling large mutant populations (Predieri 2001; Jain 2000; Jain and Maluszynski 2004). We present here a protocol for inoculating in vitro banana plants with the live agent Fusarium wilt (spores and mycelium) and for screening mutant banana seedlings under in vitro conditions.

2 Materials

2.1 Plant Tissue Culture Medium

  1. 1.

    Sterile tubes (50 ml).

  2. 2.

    Analytical balance.

  3. 3.

    Spatula.

  4. 4.

    Culture jar.

  5. 5.

    Thiamine hydrochloride.

  6. 6.

    Pyridoxine hydrochloride.

  7. 7.

    Nicotinic acid.

  8. 8.

    Myo-inositol.

  9. 9.

    6-Benzyl amino purine (BAP).

  10. 10.

    Sucrose.

  11. 11.

    Murashige and Skoog basal salt (MS) (Murashige and Skoog 1962).

  12. 12.

    Tissue culture grade water.

  13. 13.

    Gelling agent (e.g. Gelrite).

  14. 14.

    Tween 20.

  15. 15.

    Ethanol.

  16. 16.

    Sodium hydroxide (NaOH).

  17. 17.

    Sodium hypochlorite.

  18. 18.

    Magnetic stir bar.

  19. 19.

    Hot plate.

  20. 20.

    pH meter.

  21. 21.

    Erlenmeyer flasks.

2.2 Culture Media for the Isolation and Culture of Fusarium oxysporum

  1. 1.

    Petri plates.

  2. 2.

    0.22 μm Cellulose Acetate (CA) filter.

  3. 3.

    Whatman filter (pore size 8 μm).

  4. 4.

    Potatoes.

  5. 5.

    Dextrose.

  6. 6.

    Agar.

  7. 7.

    Streptomycin (or other suitable antibiotic).

  8. 8.

    Hole-puncher.

  9. 9.

    Microscope.

  10. 10.

    Haemocytometer slide.

2.3 Biological Materials

  1. 1.

    Healthy banana suckers.

  2. 2.

    Fusarium oxysporum isolate.

2.4 Gamma Irradiation

  1. 1.

    Gamma Cell with 60Co (here Gamma cell PX-30 was used).

  2. 2.

    High quality, disease-free in vitro banana plantlets.

3 Method

3.1 Preparation of Micropropagation and Rooting Medium

  1. 1.

    Prepare stock solutions (1000X or 1 mg/ml) of Thiamine, BAP, Nicotinic Acid, Pyridoxine (see Note 1).

  2. 2.

    Prepare stock solution of macro (10X concentrate) and micro (100X concentrate) elements of MS basal salt (MS medium).

  3. 3.

    For 1 l of the liquid micropropagation media, use the following amounts: 100 ml of MS macro salts, 10 ml of a MS micro salts, 5 ml BAP, 0.1 ml of Thiamine, 0.5 ml of Nicotinic acid and Pyridoxine solutions, 100 mg Myo-inositol, and 30 g sucrose. Dissolve in double distilled water.

  4. 4.

    For 1 l rooting medium use the following amounts: 50 ml of MS macro salts, 5 ml of a MS micro salts, 2 ml BAP and 0.1 ml of NAA, 0.1 ml of Thiamine, 0.5 ml of Nicotinic acid and Pyridoxine stock solutions, 100 mg Myo-inositol, 30 g sucrose, 7 g agar, and 3 g activated charcoal. Use double distilled water.

  5. 5.

    Place the media on the mixer and let it mix properly.

  6. 6.

    Calibrate the pH meter as per manufacturer instruction.

  7. 7.

    While stirring, adjust medium to pH 5.8 using NaOH or HCl.

  8. 8.

    After adjusting pH, top up the medium by adding the distilled water to 1 l.

  9. 9.

    Dispense 20 ml of the culture medium per jar.

  10. 10.

    Autoclave for 20 min at 121 °C.

  11. 11.

    Allow the medium to cool down.

  12. 12.

    Store the medium for up to a week in a cold room or in a refrigerator with 4–8 °C.

3.2 Preparation of Streptomycin Stock

  1. 1.

    Prepare Streptomycin (10 mg/ml) stock solution.

  2. 2.

    Filter Streptomycin solution using 0.22 μm cellulose acetate filters.

  3. 3.

    Keep the solution in a cold condition (4–5 °C).

3.3 Preparation of PDA (Potato Dextrose Agar) Medium

  1. 1.

    For 1 l of PDA, use 100 g of peeled potatoes (see Note 2).

  2. 2.

    Cut the peeled potatoes into four parts.

  3. 3.

    Boil potatoes for an hour in 200 ml of distilled water and filter through eight layers of cheesecloth.

  4. 4.

    Discard the solid portion; then add 10 g of dextrose and 6–7 g of agar to the clear liquid filtrate. Adjust the solution volume to 1 l by adding distilled water.

  5. 5.

    Dissolve well and heat the solution on a stirrer or in microwave (it usually takes approx. 40–50 min on the stirrer or 3–4 min in the microwave).

  6. 6.

    Dispense 500 ml in Erlenmeyer flasks (see Note 3) and autoclave immediately (100 kPa at 121 °C for 20 min).

  7. 7.

    Let it to cool to 50 °C.

  8. 8.

    Add 1.2 ml of 10 mg/ml Streptomycin solution.

  9. 9.

    Swirled the container by hand until the solution is homogenous.

  10. 10.

    Dispense 20 ml of the media in the Petri plates.

3.4 Preparation of Solid Inoculation Medium (SIM)

  1. 1.

    For 1 l of the solid culture media (1/2 MS), use the following amounts: 50 ml of Macro (10X), 5 ml of Micro (100X) solution, 7.5 g of sucrose, and 7 g of Agar. Use double distilled water (see Note 4).

  2. 2.

    Place the media on the mixer and let it mix properly.

  3. 3.

    Calibrate the pH meter as per manufacturer instruction.

  4. 4.

    While stirring, adjust medium to pH 5.8 using NaOH or HCl.

  5. 5.

    After adjusting pH, top up the medium by adding distilled water to 1 l.

  6. 6.

    Autoclave for 20 min at 120 °C.

  7. 7.

    Allow the medium to cool down.

  8. 8.

    Dispense 20 ml of the culture medium per jar.

  9. 9.

    Store the medium for up to 1 week in a cold room (keep in refrigerator with 4–5 °C).

3.5 Preparation of Inoculum

  1. 1.

    Inoculate the Foc single-spore isolates on Petri plates containing potato dextrose agar (PDA) (see Note 5).

  2. 2.

    Incubate for 1 week at 28 °C in the dark.

  3. 3.

    Cover the surface of fungal colonies by adding 5 ml of sterile water (Fig. 4.1).

  4. 4.

    Gently probe the surface of the colony with the Pasteur pipette tip generating a mixture of conidial and hyphal fragments.

  5. 5.

    Pour the suspension into a sterile Falcon tube.

  6. 6.

    Count collected conidia (macro and micro-conidia) using a haemocytometer.

  7. 7.

    Adjust inoculum concentration to 106 conidia/ml.

  8. 8.

    Use the following formula to calculate the conidia density: N × 104 × f cell/ml, where “N” is the total counted cells and “f” is dilution factor (see Note 6).

Fig. 4.1
figure 1

Preparation of the inoculum (a) Fusarium oxysporum colony (b) collection of conidia. (c) Counting cells in a haemocytometer (Source: http://insilico.ehu.eus/counting_chamber/thoma.php)

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3.6 Preparation of Filter Paper Disks

  1. 1.

    Punch the Whatman filter paper with a hole-puncher with a 5 mm diameter.

  2. 2.

    Put discs into an autoclavable container.

  3. 3.

    Autoclave twice for 20 min or one time for 40 min at 120 °C (see Note 7).

3.7 Establishment of In Vitro Cultures

  1. 1.

    Collect small and healthy suckers from a healthy plant.

  2. 2.

    Wash suckers with tap water and cut into 10 × 10 × 10 mm blocks containing shoot tips.

  3. 3.

    Surface-sterilize the shoot tips in a laminar flow cabinet with 70% Ethanol for 30 s, followed by 5% sodium hypochlorite with a few drops of Tween 20 for 30 min.

  4. 4.

    Transfer shoot tips into banana propagation liquid medium consisting of MS (Murashige and Skoog 1962) salts and vitamins, 5 mg/l BAP and 30 g/l sucrose.

  5. 5.

    Incubate shoot tips in a culture room (25 ± 1 °C, 16/8 day/night, 45–60 μmol/m2/s light intensity).

  6. 6.

    Subculture every 30 to 45 days (see Note 8).

3.8 In Vitro Mutagenesis

  1. 1.

    Prepare at least 800 shoot tips from established in vitro banana plantlets.

  2. 2.

    Transfer shoot tips into sterile Petri dishes.

  3. 3.

    Add a few drops of sterile water and seal with parafilm.

  4. 4.

    Irradiate using a suitable dose (see Note 9).

  5. 5.

    Incubate shoot tips in liquid media for 45 days at 25 ± 1 °C and 16/8 day/night.

  6. 6.

    Subculture at least three times for chimera dissolving. After the first subculture, give each plantlet a unique code to keep track of the mutant pedigree.

  7. 7.

    Separate each M1V3 propagated individual into two parts. Place one part of plantlet from each shoot onto the same propagation media, and the second half place on the rooting media (see Fig. 4.2). These plantlets are M1V4 (see Note 11).

  8. 8.

    Select healthy and well rooted mutant plantlets and transfer into the SIM media.

Fig. 4.2
figure 2

Prepare the mutant population for in vitro bioassay by pedigree method, each genotype is evaluated for disease resistance, while the same genotype is being kept for subsequent studies, as well as for the reproduction of putative resistant mutants

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3.9 In Vitro Inoculation

  1. 1.

    Select plantlets that meet the required criteria (see Fig. 4.3 and Note 12).

  2. 2.

    Incubate plantlets with healthy roots on SIM media for 2 weeks (see Note 13).

  3. 3.

    Soak 5 mm diameter filter paper discs in a conidial suspension.

  4. 4.

    Inoculate by putting one disc on the surface of the SIM media (see Fig. 4.4 and Note 15).

  5. 5.

    Incubate the inoculated plantlets at 25 ± 1 °C and 16/8 h day/night for 21–30 days.

Fig. 4.3
figure 3

Appropriate seedlings for in vitro evaluation of disease. Two-month-old seedlings 4–5 cm height of the pseudostem, have more than two fully expanded leaves and at least three white healthy roots

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Fig. 4.4
figure 4

Steps of inoculation: (a) selected plantlet incubated for 2 weeks on SIM media before inoculation, (b) autoclaved filter paper disks, (c) soaked filter disk in the inoculum suspension and placed on the surface of the SIM, (d) to (f) development of disease symptoms

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3.10 Disease Evaluation

  1. 1.

    Perform daily observations of Fusarium oxysporum development on the SIM medium.

  2. 2.

    Assay disease according to symptoms appearing 1 week after inoculation (see Note 20).

  3. 3.

    Rate disease severity on a scale of 0 to 6 (see Table 4.1 and Fig. 4.5).

  4. 4.

    Record the symptoms and the rate of disease severity 21 to 30 days post-inoculation.

  5. 5.

    Select all plantlets that show resistance (rate 0–2) (see Note 21).

Table 4.1 Disease severity-rating scale used to record symptoms caused by Fusarium oxysporum f.sp. cubense in banana plants (Wu et al. 2010)
Full size table
Fig. 4.5
figure 5

An example of in vitro bioassay of Fusarium oxysporum. M1V4 banana plantlets (local dwarf Cavendish CV) were used. The numbers (1 to 6) indicate disease severity scored according to Wu et al. (2010) after 21–30 days of inoculation

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4 Notes

  1. 1.

    It is convenient to prepare the concentrated stock solutions of macro-salts, micro-salts, vitamins, amino acids, hormones, etc. All stock solutions should be stored in a refrigerator and should be checked visually for contamination with microorganisms or precipitation of ingredients. A stock solution of vitamins, amino acids, and hormones should not be stored for an indefinite period and should be kept −20 °C.

  2. 2.

    For preparing PDA, 100 g peeled potatoes are needed. It is therefore recommended to weigh them after peeling. The peeled potatoes are cut into four-parts. Each section should not be too small or crushed. Starch should not enter into the final extract, and for the same reason, potatoes should not mash during boiling and filtering. A clear extract is needed for preparation of PDA.

  3. 3.

    To prevent liquids from spilling during the autoclaving process, dispense it into smaller volumes and pour it into larger containers. For example, pour 250 ml into a 500 ml container.

  4. 4.

    It is possible to utilize live pathogen for disease resistance screening in in vitro conditions. However, the in vitro conditions (higher humidity, reduced air velocity, media-rich in nutrients) favors the growth of microorganisms in general. To control the growth of the pathogen, the concentration of mineral salts, as well as the carbon source is reduced (see Sect. 3.4).

  5. 5.

    The protocol provided by Pérez Vicente et al. (2014) was used to isolate F. oxysporum and for single spore culture.

  6. 6.

    To count cells using a haemocytometer, add 15–20 μl of the cell suspension between the haemocytometer and a cover glass. Count the number of cells in all four outer squares, divide by four (see Fig. 4.1c, the cells in green will be counted). The number of cells per square × 104 = the number of cells/ml of the suspension.

  7. 7.

    It is recommended to autoclave the filter paper twice or to increase the time of autoclaving to ensure the filter paper is sterile.

  8. 8.

    During each subculturing, abnormal or contaminated explants were removed.

  9. 9.

    The shoot tips were irradiated in a 60Co gamma cell (dose rate was 0.018 Gy/s) with doses of 35 and 40 Gy. These doses were determined by the radio-sensitivity test. After irradiation, the explants were placed onto a fresh multiplication medium. Each growing shoot was separately multiplied up to M1V3.

  10. 10.

    About 6000 individual plantlets were screened using the in vitro bioassay described here. Finally, 21–30 days after inoculation, a total of 50 putative mutant plantlets were selected with scores 0–2. These were categorized as putative resistant mutants.

  11. 11.

    Plants which have been selected in the process of in vitro screening must be kept for further evaluation. It is therefore necessary to back up the mutant plant at the screening stage by keeping a clone of M1V4 shoot tip of each tested plant. Chimera usually dissolves after three subcultures so that mutant genotypes belonging to the same progeny should be uniform after M1V4.

  12. 12.

    In the process of selecting seedlings resistant to Fusarium disease the test material used for evaluation should have the same size. It is recommended to select plantlets with pseudostem length ranging between 4.5 to 5 cm. Selected plantlets should have more than two fully expanded leaves and at least three white roots.

  13. 13.

    Two weeks before inoculation, uniform banana plantlets should be transferred to SIM media. Plantlets under the selection must be healthy. Any stress may affect the result of pathogen screening.

  14. 14.

    Young plantlets with small size are very susceptible to the disease. The best size of the plantlet is 4–5 cm, with 2–4 leaves and roots.

  15. 15.

    The paper disk should be put next to the plantlet. Putting the inoculation source near (less than 1 cm) to the plantlet leads the pathogen to reach the seedlings faster.

  16. 16.

    During the TR4 screening process care should be taken not to spread the disease. Therefore, all containers, media, and infected plantlets should be autoclaved after the termination of the bioassay.

  17. 17.

    While preparing the inoculum suspension, care should be taken that the solution does not get contaminated with bacteria. Adding antibiotics to the inoculum solution is a way to prevent such infection. Streptomycin or Chloramphenicol is suitable for this purpose.

  18. 18.

    This protocol was developed as a fast and cost-effective method for early mass screening. It enables reduction of mutant population and identification of putative mutants for further evaluation. The in vitro resistant plantlets should be evaluated under the field conditions for confirmation of their resistance/tolerance.

  19. 19.

    To enhance root formation, it is recommended that banana plantlets are transferred to the rooting medium. However even without this step, the plantlets with 4–5 cm height would normally produce sufficient roots.

  20. 20.

    Internal symptoms should be checked and scored in the case of plantlets with symptoms such as wilting or discoloration.

  21. 21.

    TR4 symptoms in the field and in in vitro conditions are distinct. In the field, wilting is the predominant disease symptom. By contrast, due to high humidity in the in vitro assay, wilting is less significant. Instead, the appearance of necrotic leaves is more pronounced. Therefore, for disease scoring in the in vitro bioassay, one should especially score the appearance of necrotic and discolored leaves and pseudostem.