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18. Early screening technique for Fusarium wilt resistance in banana micropropagated plants - Mak, C[38]., A.A. Mohamed[39], K.W. Liew[40], Y.W. Ho[41]


Universiti Malaya
Universiti Sains Malaysia
United Plantation Bh 50603 Kuala Lumpur
11800 Penang
36009 Teluk Intan
Perak
Malaysia

Abstract

Field evaluation of disease-tolerant banana plants in soil infested with Fusarium oxysporum f. sp. cubense (FOC) is highly effective. However, it is slow because disease expression usually takes 4-5 months; and factors affecting disease expression such as inoculum concentration, edaphic conditions, temperature and other variables are difficult to control. An alternative method of screening seedlings at the nursery stage turned out to be effective. The technique requires a double-tray set up - a perforated inner tray containing sterilized river sand to grow hardened tissue cultured (TC)-plantlets, and a larger outer containment tray for collecting surplus Hoagland nutrient solution and pathogen wash-out. The inner tray fits snugly into the outer tray, which is deeper and larger. The method was tested on meristem-derived plantlets of various sizes against a range of pathogen inoculum concentrations of FOC race 4 under greenhouse conditions. Banana clones tested were 'Intan' (Pisang Berangan, AAA), 'Gold Finger' (AAAB), 'Novaria' (Cavendish, AAA), and 'Mutiara' (an improved Pisang Rastali, AAB).

Two-month-old plantlets (10-15 cm tall) were suitable for differential disease symptom expression, and gave similar results to field evaluations at the Fusarium 'hot spot'. Seedlings shorter than 10 cm did not produce consistent symptoms. Test plantlets with vigorous root systems were gently removed from the growth tray and their roots were immersed in microconidial suspensions with a wide range of concentrations in spores/ml such as 5 × 10, 5 × 102, 5 × 103, 5 × 104, 5 × 105 and 5 × 106. Two hours root immersion in the pathogen suspension was effective. All susceptible plants produced foliage and rhizome symptoms at inoculum concentrations higher than 5 × 102 spores per millilitre. Both 'Gold Finger' and 'Mutiara' showed high tolerance to Fusarium wilt, while 'Intan' and 'Novaria' were susceptible. All susceptible plants (i.e. 'Intan' and 'Novaria') showed both foliage and rhizome symptoms within 10-28 days. Over 94% of the tolerant plants and all the uninoculated control plants showed no symptoms.

The double-tray technique, besides being a rapid method for early screening against Fusarium wilt disease in banana at the seedling stage, effectively contains the disease. It eliminates cross-contamination and allows investigations on concurrent virulence testing of multiple FOC isolates against a range of test cultivars. It is readily adapted for growth-chamber studies on the effects of various environmental factors and treatments for disease expression.

1. INTRODUCTION

Fusarium wilt of bananas, caused by F. Oxysporum f. sp. cubense (FOC), is one of the most devastating diseases of banana in the tropics. It almost destroyed the banana export trade of 'Gros Michel' during the 1940s and 1950s in Latin America as a result the high susceptibility of this cultivar to race 1 of Fusarium [1]. Development of the resistant Cavendish group of cultivars provided effective and economical control, but the emergence of race 4 and its dissemination poses an immediate threat. Other attempted control measures such as injection of chemicals, soil treatments including fumigation and incorporating soil ameliorants/amendments may reduce the severity of the disease, but none of them is commercially applicable [2]. Pegg et al. [3] reported that fungicides, fumigants, flood fallowing, crop rotation, and organic amendments have rarely provided long-term control in any production area. It is therefore logical to select varieties resistant to Fusarium wilt to overcome this problem.

Field evaluation is the most reliable method for screening disease-resistant lines, but both manpower and space requirements add to the cost of screening [3]. There is also a need to maintain strict quarantine to avoid pathogen spread. In addition, plants tend not to show disease symptoms until after 4-5 months [4]. The uneven distribution of pathogen in the field can lead to 'disease escapees' while many variables that can affect infection and symptom expression cannot be altered nor controlled (Figure 1).

Figure 1 Field screening in Fusarium 'hot spot'

Earlier researchers expressed the need for improved methods of testing small plants, not only for screening for resistance but also for comparative virulence and pathogenicity studies [5,3]. The first report on small-plant testing indicated that roots of banana seedlings were inoculated with fungal spores before transfer to an infested field [6]. Susceptible banana plantlets showed external symptoms of leaf yellowing within 2 weeks and wilted within 4 weeks of inoculation [7]. Two-month-old seedlings of M. balbisiana and meriplants with 6-8 leaves gave reproducible results that were very similar to the field response [8].

Earlier, a double-cup sand-culture containment method had been developed for testing pathogen virulence [9]. 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 so as to eliminate cross-contamination. It 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. The present paper describes the methodology, and verification of the reliability of the technique by considering several factors, including concentrations and duration of pathogen inoculation, age of host plants, and ability to show differential responses similar to field evaluations.

2. MATERIALS AND METHODS

2.1. Planting materials

Several years of field experiments in a Fusarium wilt infected 'hot spot' have shown that 'Intan' (an improved Pisang Berangan (AAA) and 'Novaria' (early maturing Dwarf Cavendish, AAA) are susceptible, but 'Mutiara' (an improved Pisang Rastali, AAB) and 'Gold Finger' (FHIA 1, AAAB) are tolerant to FOC race 4. These cultivars were selected as susceptible and tolerant testers in the present study. Suckers of these cultivars were micropropagated by using shoot-tip meristem culture.

2.2. Inoculum and plantlet preparation

Tissue culture-derived plantlets were transferred to the double-container apparatus for hardening, as well as to grow them in the greenhouse until they attained the desired size. The double compartment consists of a tray measuring 43 × 29 × 9 cm, which fits snugly into another, larger, outer tray measuring 46 × 31 × 20 cm (Figure 2). The upper tray was three-quarters-filled with sterilized fine river sand. The plantlets were watered with tap water every other day, plus Hoagland's solution once a week. After 45 days of acclimatisation, the test plantlets were carefully uprooted, and only those with healthy white roots were selected for inoculation by immersion in the appropriate conidia suspension for 2 h before tagging and replanting in the trays for maintenance and observation in the greenhouse. Plantlets were watered using Hoagland's Complete Nutrient Solution [10].

Figure 2 The Double Tray System

Race 4 of Fusarium oxysporum f. sp. cubense was freshly isolated from susceptible 'Novaria' plants on Potato Dextrose Agar [11] followed by single spore isolation [12] and inoculum preparation in Armstrong's Liquid Medium [11]. Cultures incubated at room temperature and shaken twice a day for 7 days were filtered through two layers of cheesecloth. The desired inoculum concentrations of microconidia were prepared using a haemocytometer, and roots were inoculated immediately.

2.3. Evaluation

Leaf symptoms on susceptible plants were observed within 10-14 days of inoculation. The numbers of leaves showing disease symptoms were recorded after the first 2 weeks and again after 4 weeks. Final evaluation in the fifth week was based on the Leaf Symptom Index (LSI) (Figure 3) and Rhizome Discoloration Index (RDI) (Figure 4) [13]. A slight change was made in the RDI by the addition of an eighth point on the scale to differentiate dead plants from those showing 100% rhizome discoloration.

Figure 3 Scales of Leaf Symptom Index (LSI)

1 No streaking or yellowing of leaves. Plant appears healthy
2 Slight streaking and/or yellowing of lower leaves.
3 3 Streaking and/or yellowing of most of the lower leaves.
4 Extensive streaking and/or yellowing on most or all of the leaves.
5 Dead plant.

Figure 4 Scales of Rhizome Discoloration Index (RDI)

1 No discoloration of tissue of stellar region of rhizome or surrounding tissue.
2 No discoloration of stellar region of rhizome; discoloration at junction of root and rhizome.
3 Trace to 5% of stellar region discolored.
4 6-20% of stellar region discolored.
5 21-50% of stellar region discolored.
6 More than 50% of stellar region discolored.
7 Discoloration of the entire rhizome stele.
8 Dead plant.

After recording LSI and RDI, the overall Disease Severity Index (DSI) for leaf symptoms and rhizome discoloration for each treatment was calculated as follows:

The DSI consists of four designations, namely resistant, tolerant, susceptible and highly susceptible (Table 1). If the cultivar is resistant in LSI and tolerant in RDI, the cultivar is considered as tolerant. If RDI is tolerant and LSI is susceptible, the cultivar is susceptible. The final status of the cultivar is considered as resistant when both LSI and RDI for each treatment show resistance. If one of the responses is tolerant, the cultivar is then considered as tolerant.

Table 1 Translation of DSI scales

DSI Scales for LSI

DSI Scales for RDI

Translation

1

1

Resistant

Between 1.1 and 2

Between 1.1 and 3

Tolerant

Between 2.1 and 3

Between 3.1 and 5

Susceptible

Between 3.1 and 4

Between 5.1 and 8

Highly susceptible

3. RESULTS

3.1. Establishment of spore concentration suitable for disease expression

Experiments were carried out to ascertain the appropriate spore concentration, duration of spore inoculation, and age of plantlets suitable for disease expression.

3.1.1. Effect of spore concentrations on symptom expression

Nine plantlets of Pisang Berangan with different height were tested for symptom expression by treating with different concentrations of spores per millilitre, ranging from 5 × 10, through 5 × 102, 5 × 103, 5 × 104, 5 × 105, to 5 × 106. At all concentrations of spores, plantlets showed disease symptoms between 10 days to 4 weeks, except those treated with 5 × 10 spores per millilitre. Most of the plantlets treated with 5 × 102 and 5 × 103 spores per millilitre produced mild symptoms relatively late (i.e. between 3 and 4 weeks) and then progressed gradually towards severity. At higher concentrations of 5 × 104, 5 × 105 and 5 × 106, plantlets showed symptoms between 10 days and 3 weeks which became very severe at the end of the fourth week. Thus there was a gradual increase in disease severity as the spore concentration increased. A spore concentration higher than 5 × 102 should be used to ensure symptom expression.

3.1.2. Effect of inoculation duration on symptom expression

Three plantlets of Pisang Berangan were used for root immersion in medium containing 5 × 106 spores per millilitre at four different time intervals, 2 h, 2.5 h, 3 h, and 3.5 h. All plantlets produced disease symptoms although the degree of expression varied. Hence a 2 h treatment time appeared suitable for effective inoculation.

3.1.3. Effect of plant height on symptom expression

Plantlets at different stages of growth (also at different heights), namely 2-3 weeks, 5-6 weeks and 7-8 weeks, were inoculated with 5 × 106 spores per millilitre. After 3 weeks of inoculation, no disease symptom was observed in 5-7 cm tall (2-3-weeks-old) plantlets. Moderate to severe symptoms appeared in 10 cm tall (5-6-week-old) and 15 cm tall (7-8-week-old) plantlets. It is likely that vascular tissues have not yet fully developed in plantlets shorter than 10 cm, so that the pathogen cannot become established in the host; this can be achieved only when conidia are taken up through the transpiration stream in the vascular tissues [14].

3.2. Application of the 'double-tray' technique to screen for Fusarium wilt tolerance

Four banana clones ('Intan', 'Mutiara', 'Novaria' and 'Gold Finger') were tested against FOC race 4 using the double-tray method. For each clone, 30 plantlets were hardened (6-8 weeks) and grown to 10-15 cm height. Roots were immersed for 2 h in three different inoculum concentrations (spores per millilitre), 5 × 106 (high), 5 × 104 (medium) and 5 × 102 (low). The controls were immersed in sterile distilled water without spores.

All inoculated plantlets of susceptible cultivars (i.e. 'Intan' and 'Novaria') expressed visible disease symptoms after 10-14 days in the field. Leaf chlorosis appeared first in the older leaves and then progressed upwards to the younger leaves. More than half of the inoculated 'Intan' plantlets remained healthy when treated with a concentration of 102 spores per millilitre of inoculum. However, plants inoculated with higher spore inoculum concentrations (104 and 106 spores per millilitre) showed severe leaf and rhizome symptoms. 'Intan' was designated as susceptible based on overall DSI status. For 'Novaria', 13 of the 30 seedlings inoculated with the low spore concentration of the pathogen did not show leaf symptoms. However, ten of them showed varying degrees of rhizome discoloration. The degree of leaf symptom expression and rhizome discoloration intensified with increasing spore concentrations, and only one plant did not show both leaf symptoms and rhizome discoloration at a concentration of 106. The cultivar was considered to be highly susceptible to race 4.

The field tolerant cultivars, namely 'Mutiara' and 'Gold Finger', did not produce external symptoms and remained healthy until the fifth week, when most of the susceptible cultivars were either dying or showed severe external symptoms. Only five out of the 90 'Mutiara' plantlets that were inoculated with a high spore concentration showed symptoms on the older leaves by the third week. Most of the plantlets without external leaf symptoms had no rhizome discoloration. 'Gold Finger' showed good tolerance at the three spore concentrations used. None of the inoculated plantlets produced leaf symptoms; however, about 50% of the inoculated plants produced slight rhizome discoloration, which were limited to small areas at root junctions. The final status of 'Gold Finger' was considered to be tolerant.

The pathogen was recovered from infected plant parts. None of the control seedlings showed any symptoms. The DSI for both LSI and RDI of the four cultivars and their final status are summarized in Tables 2 and 3. These results are in agreement with those obtained from field evaluation in the Fusarium 'hot spot' screening.

Differences in disease expressions were observed in plantlets of the same cultivar inoculated with the same spore inoculum concentration. This indicated that disease expression might still be influenced by factors not considered in the experiments, such as light and temperature. In addition, somaclonal variation might arise during tissue culture [2]. Perhaps resistance to Fusarium wilt disease is a polygenic trait [15].

Table 2 Response of four banana cultivars to Fusarium wilt pathogen at 5 weeks after inoculation

Cultivars

Inoculum concentration (spores/ml × 5)

Number of inoculated plants

Number of infected plants

DSI

Status of disease expression

LSI

RDI

Intan

102

30

12

1.7

2.2

Tolerant

Intan

104

30

24

2.7

4.4

Susceptible

Intan

106

30

28

3.2

5.8

Highly susceptible

Mutiara

102

30

0

1

1

Resistant

Mutiara

104

30

3

1.1

1.1

Tolerant

Mutiara

106

30

2

1.1

1.4

Tolerant

Novaria

102

30

25

1.8

4.6

Susceptible

Novaria

104

30

27

2.7

5.5

Highly susceptible

Novaria

106

30

29

2.6

5.6

Highly susceptible

Gold Finger

102

30

19

1

2.9

Tolerant

Gold Finger

104

30

7

1

1.6

Tolerant

Gold Finger

106

30

17

1

1.9

Tolerant

4. DISCUSSION

The most effective way of controlling Fusarium wilt disease is by developing resistant host plants. Screening of somaclonal variants in Fusarium infested soil or 'hot spots' has been useful in selecting tolerant plants [10,16]. In general, disease expression was observed in 4-5-month-old plants. However, there are problems of quarantine control to avoid disease spread, field escape due to uneven pathogen distribution, and influence of environmental and soil variables.

Table 3 The overall status of disease expression of four banana cultivars

Cultivar

Spore concentrations during inoculation

Final status

Low

Medium

High

Intan

Tolerant

Susceptible

Highly susceptible

Highly susceptible

Novaria

Susceptible

Highly susceptible

Highly susceptible

Highly susceptible

Mutiara

Resistant

Tolerant

Tolerant

Tolerant

Gold Finger

Tolerant

Tolerant

Tolerant

Tolerant

Inoculation of young seedlings at the nursery stage can produce severe symptoms that are not expressed in the field [3]. However, we found that the cultivars that were tolerant in the field were also tolerant when tested using the double-tray technique. Stover and Buddenhagen [17] questioned the durability of resistance shown by small plants inoculated in flats when transferred to a disease-infected field. We took plantlets that had survived the pathogen infection in the double-tray and planted them in the Fusarium 'hot spot' field. These plants were still tolerant after one year in the field. The double compartment or 'double-tray' technique' was adequate for differentiating tolerant (Mutiara and Gold Finger) and susceptible (Intan and Novaria) varieties. The advantages of the double compartment technique are:

(a) Containment of pathogen was adequate using the snugly fitting outer tray.

(b) Easy sterilization of contaminated materials. Soil is sterilized in the autoclave. Trays can be washed with a solution of sodium hypochlorite.

(c) Portable experimental set-up including soil, trays and seedlings enables screening under predetermined environmental conditions.

(d) Ability of hardened plantlets, 2 months old (10-15 cm tall), to exhibit differential host response to pathogen challenge.

(e) Reduction in time for disease symptoms to appear (10-30 days instead of 120-150 days needed in the field evaluation).

(f) Small space requirement - a single set of trays (43 × 29 × 9 cm tray on a larger outer tray of 46 × 31 × 20 cm) can accommodate up to 20 plantlets.

(g) Root immersion inoculation provides an effective challenge and eliminates the possibility of disease escape.

REFERENCES

[1] PEGG, K.G., LANGDON, P.W., "Fusarium wilt (Panama Disease)- a review", Banana and Plantain Breeding Strategies, (PERSLEY, G.J., DE LANGHE, E.A., Eds), ACIAR Proceedings No. 21 (1987) 119-123.

[2] HWANG, S., KO, W.H., "Somaclonal variation of bananas and screening for resistance to Fusarium wilt", Banana and Plantain Breeding Strategies. (PERSLEY, G.J., DE LANGHE, E.A., Eds), ACIAR Proceedings. No. 21. (1987) 151-156.

[3] PEGG, K.G., et al., Fusarium wilt of banana in Australia: a review. Aust. J. Agr. Res. 47 (1996) 637-650.

[4] MORPURGO, R., et al., Selection parameters for resistance to Fusarium oxysporum f. sp. cubense race 1 and race 4 on diploid banana (Musa acuminata Colla), Euphytica 75 (1994) 121-129.

[5] BUDDENHAGEN, I.W., "Disease susceptibility and Genetics in relation to breeding of banana and plantains", Banana and Plantain Breeding Strategies. (PERSLEY, G.J., DE LANGHE, E.A., Eds), ACIAR Proceedings No. 21 (1987) 95-109.

[6] VAKILI, N.G., Fusarium wilt resistance in seedlings and mature plants of Musa species, Phytopathology 55 (1965) 135-140.

[7] SUN, E.J., SU, H.J., Rapid method for determining differential pathogenicity of Fusarium oxysporum f. sp. cubense using banana plantlets, Trop. Agr. (Trinidad) 61 (1984) 7-8.

[8] EPP, M.D., "Somaclonal variation in banana: a case study with Fusarium wilt", Banana and Plantain Breeding Strategies, (PERSLEY, G.J., DE LANGHE, E.A., Eds), ACIAR Proceedings No. 21 (1987) 140-150.

[9] LIEW, K.W., "Screening for disease resistance in banana plantlets against Fusarium wilt", (Paper presented at the Regional Training Course on Molecular Approaches, Mutation and other Biotechnologies for the Improvement of Vegetatively Propagated Plants (FAO-UKM), 28 Oct-8 Nov. 1996, Bangi, Selangor, Malaysia) (1996).

[10] EPSTEIN, E., Mineral nutrition of plants: principles and perspectives. John Wiley and Sons, New York (1972).

[11] SINGLETON, L.L. et al., "Appendix", Methods for Research on Soilborne Phytopathogenic Fungi. (SINGLETON, L.L., et al., Eds), APS Press, St. Paul, Minnesota. (1992).

[12] BOOTH, C., Fusarium: Laboratory guide to the identification of the major species. Commonwealth Mycological Institute, Kew, UK (1977).

[13] BRAKE, V., et al., The influence of temperature, inoculum level and race of Fusarium oxysporum f. sp. cubense on the disease reaction of banana cv. Cavendish, Aust. J. Agr. Res. 46 (1995) 673-685.

[14] BECKMAN, C.H., "Host responses to the pathogen", Fusarium wilt of Banana. (PLOETZ, R.C., Ed.), APS Press. St. Paul, Minnesota (1990) 93-105.

[15] ORTIZ, R., "Musa genetics", Bananas and Plantains, (GOWEN, S., Ed.), Chapman & Hall, London (1995) 84-109.

[16] HO, Y.W., "The development of Pisang Mutiara - a Fusarium wilt tolerant Rastali", Proceedings of the First National Banana Seminar, (WAHAB. Z., et al., Eds), (1999) 44-47.

[17] STOVER, R.H., BUDDENHAGEN, I.W., Banana breeding: polyploidy, disease resistance and productivity, Fruits 41 (1986) 175-191.


[38] Institute of Biological Science
[39] Institute of Biological Science
[40] School of Biological Sciences
[41] Research Department

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