Keywords

1 Introduction

Banana is the second most commonly grown fruit crop in Malaysia. About 50% of the banana growing area is cultivated with Pisang Berangan and the Cavendish type. However, in the recent years, the overall banana production and cultivation areas have decreased due to the increasing threat of Fusarium wilt and Moko diseases. This alarming issue has led to increasing prices and limited availabilities of banana fruits for local consumption and export (MOA report 2017).

Fusarium wilt of bananas, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases of banana in most parts of the world including Malaysia. This severe disease almost crippled the world banana plantation, production and export trade of ‘Gros Michel’ circa 1940s and 1950s due to high susceptibility of this cultivar to Fusarium Race 1 (Pegg and Langdon 1987). Introduction of the resistant Cavendish group of cultivars provided an effective and economical solution but the emergence of Foc TR4 and its dissemination in the tropics and sub-tropics poses an immediate threat (Asif and Mak 2001). In Malaysia, we have experienced a total wipe out of a commercial scale Cavendish plantation in the southern part of Malaysia during the 1990s due to the destructive Foc TR4 disease.

Various control measures such as injection of chemicals, soil treatments including fumigation and incorporating soil amendments may reduce the severity of the disease, but none of them is commercially applicable (Hwang and Ko 1987). It was also reported that fungicides, fumigants, flood fallowing, crop rotation, and organic amendments have rarely provided long-term control in any production area (Pegg et al. 1996). Among three races of Foc namely Race 1, Race 2 and Tropical Race 4 which attack bananas, Tropical Race 4 is the most pathogenic and affects many banana cultivars including Cavendish. The pathogen, Foc, can be disseminated through suckers, soil, water, and by farming practices when farmers use contaminated tools. Chemical control, such as soil fumigation, is a promising measure but is hazardous to the environment. The pathogen persists in the contaminated soil by producing chlamydospores even in the absence of the host bananas or sometimes by infection of roots of some weeds (Pegg et al. 1996). As a result, once the field is invaded by Foc, the field cannot be used for banana production for up to 30 years (Asif and Mak 2001). It is therefore of high importance to develop and select new banana varieties that are resistant to Fusarium wilt to overcome this problem (Rusli 2011). One of the techniques to produce new varieties is through in vitro mutagenesis with the application of gamma irradiation (Mak et al. 1995). Gamma irradiation is a reliable and popular physical mutagen to increase genetic diversity and induce mutations and has led to the establishment of new plant varieties (Norazlina et al. 2014).

Field evaluation is the most reliable method for screening disease-resistant lines, but both manpower and space requirements are the limitations that add to the cost of screening (Pegg et al. 1996). It is also essential to maintain strict quarantine measures to avoid pathogen spread and cross-contamination. In addition, plants tend not to show disease symptoms until after 4–5 months (Morpurgo et al. 1994). The uneven distribution of the pathogen in the field can lead to ‘disease escape’ while many variables that can affect infection and symptom expression cannot be altered nor controlled (Asif and Mak 2001). Alternatively, methods that are simpler, cost effective, low maintenance with space requirements that can show early detection against Fusarium wilt are more attractive and have been developed by many laboratories.

Earlier studies reported the need for improved methods like pre-screening or early detection of tolerance against Fusarium wilt using ex vitro or in vitro rooted plantlets. This early detection is not only for screening for tolerance/resistance but also for comparative virulence and pathogenicity studies (Buddenhagen 1987; Pegg and Langdon 1987). This early detection can be carried out at lab-based or nursey-based stages prior to field-screening.

An earlier study reported roots inoculated by dipping or soaking with a fungal spore suspension before being transferred to an infested field (Mak et al. 2001; Vakili 1965). Susceptible banana plantlets showed external symptoms of leaf yellowing within 2 weeks and wilted within 4 weeks of inoculation (Vakili 1965). Earlier, a double-cup sand-culture containment method had been developed for testing pathogen virulence (Liew 1996). Modification was done and it was replaced by a ‘double compartment’ apparatus, which contains two plastic trays, one fitting inside the other. This double-tray technique has the capacity for pathogen containment to eliminate cross-contamination (Mak et al. 2001). This technique can be further modified to investigate the effects of various inoculum concentrations and environment variables on infection and disease expression. A bigger size tray could screen a greater number of plants. However, this system must be done in a nursery that still requires a large space with special containment, manpower for the preparation of the system and the cost for the set-up and modification is also less effective.

In this study, a lab-based screening using hydroponic system has been developed. This system is simpler, soilless, and easy to set up, cost effective, portable, requires less space and maintenance. The system can be modified to suit the requirements of the work in the laboratory. In this chapter, protocols on acclimatization of ex vitro irradiated rooted plantlets, inoculation with Foc TR4 conidial suspension, lab- based screening using hydroponic system and observation for early detection of disease symptoms and scoring of disease severity are presented. This work also describes the methodology, reliability of this lab-based screening by considering several factors, including the concentration and duration of Foc TR4 conidial suspension inoculation, type of host plants, and the ability to show differential disease symptoms similar to nursery-based and field evaluations.

2 Materials

2.1 Acclimatization of Banana Plantlets

  1. 1.

    Irradiated banana cv. Berangan (AAA) in vitro rooted plantlets with the pseudostem height of 5–7 cm (see Notes 1 and 2).

  2. 2.

    Ventilated culture containers (see Note 5).

  3. 3.

    Perlite (see Note 6).

  4. 4.

    Forceps.

2.2 Fusarium oxysporum f. sp. cubense (Foc) Tropical Race 4 (TR4) Cultures

  1. 1.

    Potato dextrose agar (PDA) medium.

  2. 2.

    1% yeast extract.

  3. 3.

    Foc TR4 stock culture (see Note 7).

2.3 Foc Conidial Suspension

  1. 1.

    Sterile distilled water.

  2. 2.

    Sterile glass rod.

  3. 3.

    Filter funnel.

  4. 4.

    Cotton wools.

  5. 5.

    500 ml glass beaker.

  6. 6.

    Haemocytometer.

2.4 Soaking in Foc TR4 Conidial Suspension

  1. 1.

    Foc TR4 suspension (106 spores/ml).

  2. 2.

    Ex vitro irradiated cv. Berangan rooted plantlets (10–15 cm pseudostem height) (see Note 1).

  3. 3.

    Soaking containers.

  4. 4.

    Biosafety Cabinet Level 2 (see Note 8).

2.5 Screening in Hydroponic System

  1. 1.

    Hydroponic system (see Note 10).

  2. 2.

    Distilled water.

  3. 3.

    Foc inoculated plantlets.

  4. 4.

    Nutrient rich solution (see Note 11).

3 Methods

3.1 Preparation of Acclimatized Ex Vitro Plantlets

  1. 1.

    Select healthy, contamination-free in vitro rooted plantlets for acclimatization phase (see Fig. 6.1).

  2. 2.

    Gently remove in vitro rooted plantlets with the pseudostem height of 5–7 cm from rooting media (see Fig. 6.1, Note 3).

  3. 3.

    Wash under running tap water to remove any traces of adhering agar/media.

  4. 4.

    Transfer plantlets using forceps into ventilated culture containers containing perlite. Each ventilated container can be filled with 12–15 plantlets (see Fig. 6.1).

  5. 5.

    Place the ventilated culture containers in hardening room at room temperature for 3 weeks (see Fig. 6.2).

  6. 6.

    After 3 weeks, ex vitro rooted plantlets with pseudostem height of 10–15 cm are ready to be advanced to lab-based screening protocol using hydroponic system (see Fig. 6.2).

Fig. 6.1
figure 1

Berangan in vitro rooted plantlets selected for acclimatization. (a) Rooted Berangan cultures in rooting media. (b) In vitro rooted plantlets with the pseudostem height of 5–7 cm. (c) Washing and removal of adhering agar/media from in vitro rooted plantlets. (d) Transfer of plant material into ventilated culture containers containing perlite

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Fig. 6.2
figure 2

Acclimatization of Berangan ex vitro rooted plantlets. (a) Ventilated culture containers placed in hardening room. (b) 3-weeks old acclimatized Berangan rooted plantlets. (c) Berangan rooted plantlets with pseudostem height of 10–15 cm ready to be advanced to lab-based screening protocol using hydroponic system

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3.2 Subculturing Foc TR4

  1. 1.

    Subculture Foc TR4 onto fresh PDA medium by taking one agar slab (0.5 × 0.5 cm) containing the fungal mycelium (see Fig. 6.3).

  2. 2.

    Transfer agar slab onto PDA medium supplemented with 1% yeast extract for further multiplication.

  3. 3.

    Incubate cultures under continuous light at 23–25 °C in growth chamber.

  4. 4.

    Observe the growth of Foc mycelium. Growth usually will take about 5–7 days (see Fig. 6.3).

Fig. 6.3
figure 3

Foc TR4 cultures. (a) Subculture of Foc TR4. (b) One agar slab (0.5 × 0.5 cm) cut. (c) Growth of Foc mycelium onto PDA medium

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3.3 Preparation of Foc Conidial Suspension

  1. 1.

    Pour 10 ml of sterile distilled water onto PDA plates containing Foc mycelium and scrape all mycelium using a sterile glass rod.

  2. 2.

    Set up a filter funnel with plugging cotton wools on the mouth of a 500 ml glass beaker.

  3. 3.

    Pour the suspension through the funnel to separate hyphae from spores in the filtrate.

  4. 4.

    Using a haemocytometer count the number of spores/ml.

  5. 5.

    Dilute the suspension into desired concentrations (106 spores/ml) using sterile distilled water (see Fig. 6.4).

Fig. 6.4
figure 4

Foc TR4 suspension (106 spores/ml) ready to be used for lab-based screening

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3.4 Soaking of Rooted Plantlets in Foc TR4 Conidia Suspension (106 Spore/ml)

  1. 1.

    Remove ex vitro rooted plantlets with pseudostem height of 10–15 cm after 3 weeks acclimatization in perlite (see Fig. 6.5).

  2. 2.

    Wash roots under running tap water to remove any traces of adhering perlite (see Fig. 6.5).

  3. 3.

    Ex vitro rooted plantlets with pseudostem height of 10–15 cm ready for pathogenicity test (see Note 13).

  4. 4.

    Inoculate root by soaking in the Foc TR4 conidia suspension (106 spore/ml) (see Fig. 6.6).

  5. 5.

    Conduct 3 treatments with different soaking periods for pathogenicity test (see Fig. 6.6):

    1. (a)

      T0: Control (non-inoculated with Foc TR4);

    2. (b)

      T1: 106 spore/ml Fusarium solution for 1 h;

    3. (c)

      T2: 106 spore/ml Fusarium solution for 2 h;

    4. (d)

      T3: 106 spore/ml Fusarium solution for 3 h.

Fig. 6.5
figure 5

Acclimatized rooted plantlets ready to be soaked in Foc TR4 conidial suspension. (a) Ex vitro rooted plantlets removed from perlite. (b) Washing roots to remove adhering perlite. (c) Rooted plantlets with pseudostem height of 10–15 cm ready for pathogenicity test

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Fig. 6.6
figure 6

Inoculation of roots with Foc TR4. (a) Soaking the irradiated rooted plantlets with Foc TR4 conidia suspension (106 spore/ml). (b) Soaking with different intervals (hours) under biosafety cabinet level 2

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3.5 Screening in Hydroponic System

  1. 1.

    Fill the hydroponic system with distilled water. In this type of hydroponic system, 15 l of water is used (see Fig. 6.7).

  2. 2.

    Gently transfer inoculated plantlets from all treatments into thumb-pots of the hydroponic system for screening of Foc TR4 disease symptoms (see Fig. 6.7).

  3. 3.

    Screening for early detection against Foc TR4 is to be conducted in hardening room under natural conditions (see Fig. 6.8).

  4. 4.

    Symptom observations should be conducted weekly.

Fig. 6.7
figure 7

Screening in hydroponic system. (a) Fill 15 l water in the lower compartment of the hydroponic system. (b) Transfer inoculated plantlets into thumb-pots of the hydroponic system

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Fig. 6.8
figure 8

Screen for early detection of resistance against Foc TR4

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3.6 Observation of Disease Symptoms

  1. 1.

    Conduct weekly observations for disease development (28 days period).

  2. 2.

    Visual screening and observation of disease symptoms on weekly basis.

  3. 3.

    Rate disease severity on a scale of 0 to 6 (Wu et al. 2010) (see Table 6.1).

  4. 4.

    Select the workable and efficient treatment amongst the three different periods.

  5. 5.

    Proceed with the chosen treatment for further lab-based screening using a bigger population of inoculated plantlets (preferably 1000–2000) (see Note 14).

Table 6.1 Disease severity scale (Wu et al. 2010)
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3.7 Observation and Results from Lab-Based Screening Using Hydroponic System

  1. 1.

    After 28 days of inoculation with Foc TR4 (106 spore/ml) with different inoculation periods (1 h, 2 h, 3 h), conduct scoring of disease symptoms (see Fig. 6.9b).

  2. 2.

    Dissect plantlets from all treatments for scoring/rating (see Fig. 6.9c).

  3. 3.

    For treatment 0 (control), the irradiated plantlets that were not inoculated with Foc conidial suspension showed no disease symptoms and these plantlets developed into healthy seedlings (see Fig. 6.9d).

  4. 4.

    Rate degrees of disease symptoms in T1 (1 h soaking period), T2 (2 h soaking period) and T3 (3 h soaking period) in Foc suspensions (Fig. 6.10).

  5. 5.

    Observe discoloration of pseudostem of the treated plantlets (see Fig. 6.9e–g).

  6. 6.

    Score plantlets from all treatments based on leaf symptoms responses (Brake et al. 1995) (see Fig. 6.11).

  7. 7.

    Banana plantlets showed external symptoms of leaf yellowing within 10–14 days.

  8. 8.

    The surviving plants showed disease scoring of 1–2.

    1. (a)

      Score 1: No streaking or yellowing of leaves. Plant appears healthy.

    2. (b)

      Score 2: Slight streaking and/or yellowing of lower leaves.

Fig. 6.9
figure 9

Disease scoring (Wu et al. 2010). (a) Rooted plantlets after 28 days of inoculation. (b) Conduct scoring of disease symptoms for all treatments. (c) Plantlets cut-open for scoring. (d) Corm completely clean, no vascular discoloration (control). (e) ≤1/2 the height of the pseudostem was discolored (T3). (f) >1/2 the height of the pseudostem was discolored and (or) there was discoloration of the leaf stalk (T1). (g) Total discoloration of vascular tissue (T2)

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Fig. 6.10
figure 10

Average severity index and average plant height for different inoculation time based on the disease scoring (Wu et al. 2010). Severity at the scale of 1 to 2 for T1 and T2. Average severity index was at 0.50 for treatment 1 and 0.88 for treatment 2. Scoring of 0 to 1 was observed in T3 treatment with average severity index of 0.13. Plant development (height) was decreased for both T1 and T2 treatment, with an average of 9.72 cm as compared to the non-inoculated plantlets; 13.63 cm

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Fig. 6.11
figure 11

Disease scoring observation on leaves symptomatic responses after inoculation in Foc suspension. (a) Non-inoculated plantlets showed no leaves yellowing. (b) T1 – 1 h soaking period. (c) T2 – 2 h soaking period. (b) T3 – 3 h soaking period

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4 Notes

  1. 1.

    Mutagenesis of banana (cv. Berangan Intan) plantlets was performed by irradiating meristem tissues/shoot tips with acute gamma irradiation using BioBeam GM8000 (Germany) with a Caesium-137 source and chronic gamma irradiation using Gamma Greenhouse with a Caesium-137 source. The meristem pieces (about 1 cm × 2 mm) were aseptically excised from in vitro plantlets of Pisang Berangan. Each meristem was cut longitudinally into two pieces. A total of 50 meristem pieces for each dose were transferred into sterile moist Petri dishes and sealed with parafilm. The meristems in the Petri dishes were irradiated with acute gamma ray using gamma cell BioBeam GM8000 and chronic gamma irradiation using Gamma Greenhouse with a Caesium-137 source at 0, 10, 20, 30, 50, 70, 90, 120 Gy. Each growing irradiated shoot from all the optimal doses (chronic irradiation: 30, 50, 70 Gy and acute irradiation: 10, 20, 30 Gy) were separately sub-cultured to M1V4 generation (three subcultures at monthly interval) to minimize chimerism.

  2. 2.

    Irradiated plantlets (after M1V4) were continuously maintained in multiplication medium, modified MS Medium (Murashige and Skoog 1962) supplemented with 2.5 mg/l BAP and 0.1 mg/l NAA for further proliferation and plantlets multiplication.

  3. 3.

    For root induction, in vitro plantlets with the pseudostem height of 3–5 cm were cultured in MS medium supplemented with 1 mg/l IBA + 0.1% activated charcoal. Cultures were incubated at 24 °C with a 16 hours photoperiod (3500 lux).

  4. 4.

    All in vitro experiments should be conducted in a dedicated Plant Tissue Culture laboratory that should have a Laminar Air Flow cabinet and must adhere to proper decontamination procedures and disposal of waste.

  5. 5.

    The ventilated culture containers used during acclimatization of ex vitro rooted plantlets provide high humidity that is needed to help the plantlets to initiate the development of cuticles, stomata and root functions for better regeneration.

  6. 6.

    Perlite is used during acclimatization phase because it provides a soilless growth medium that can improve aeration, well-drained and eliminate any source of cross-contamination if soil-like growth medium is used.

  7. 7.

    The initial Foc TR4 cultures were kindly provided by the Plant Pathology Unit of Malaysian Agriculture Research and Development Institute (MARDI). The initial Foc TR4 cultures were sampled by MARDI from infected Berangan plants, the strain was tested by Koch Postulate test and were used to formulate the Foc TR4 hotspot for screening and evaluation against Foc TR4 disease. The Foc stock cultures were maintained by routine subculturing onto fresh PDA media and maintained under continuous light at 23–25 °C in a growth chamber.

  8. 8.

    All Foc TR4 related work and experiments i.e.; subculturing, preparation of conidial suspension and soaking/inoculating of roots in conidial suspension should be conducted with caution using suitable Personal Protective Equipment (PPE), dedicated labwares, dedicated biosafety cabinet level 2 in a dedicated Plant Pathology laboratory.

  9. 9.

    All labwares, glasswares, waste, disposable items related with Foc work must be separated from others and must always adhere to strict decontamination procedures and waste disposal.

  10. 10.

    There are many types of hydroponic systems for screening Foc disease symptoms. One can choose a hydroponic system that is the most suitable to work with. The principle is to ensure that the inoculated roots are totally immersed in the water.

  11. 11.

    The hydroponic nutrient rich solution contains Calcium Nitrate, Iron Chelate, Kalium Nitrate, Mono-Kalium Hydrogen Phosphate, Magnesium Sulphate, Manganase Sulphate, Boric Acid, Zinc Sulphate, Copper Sulphate and Natrium Molybdate.

  12. 12.

    Hydroponic system eliminates cross-contamination and allows concurrent investigations of Foc isolates against a range of irradiated rooted plantlets population.

  13. 13.

    Pathogenicity experiment can be conducted as a prerequisite for the establishment of a rapid and efficient Foc TR4 lab-based screening method for early detection of disease symptoms. This experiment can test the effectiveness of the Foc TR4 suspension (106 spores/ml) to cause disease symptoms to banana plantlets. It can also optimize the Foc TR4 soaking periods (minutes, hours) and the conidial suspension concentration.

  14. 14.

    Lab-based screening on bigger population of 1000–2000 irradiated rooted plantlets is recommended. The surviving plants that showed some degree of tolerance with severity index of 1–2 (Brake et al. 1995) will be screened under nursery-based condition and later proceed to field-screening for selection of possible mutant lines.

  15. 15.

    From this study, the early screening method using hydroponic system provided baseline data on the development and optimization of an efficient and workable lab-based screening of Foc disease.

  16. 16.

    Soaking of roots of the irradiated plantlets in Foc TR4 (106 spore/ml) for 2 h interval was found to be effective.

  17. 17.

    It gave early expression of symptoms, as early as 14 days and produced consistent symptoms during the screening.

  18. 18.

    Soaking of roots of the irradiated plantlets in Foc TR4 (106 spore/ml) for 2 h and screening using hydroponic system for early detection of Foc TR4 tolerance/resistance is effective to be used for screening of a bigger population (1000–2000 irradiated plantlets).

  19. 19.

    The plantlets that survived after inoculation and showed some degree of tolerance i.e., disease scoring of 1–2 (Brake et al. 1995) are advanced to nursery-based screening before being selected as putative mutants for field-based screening at a Foc TR4 hotspot.