Department of Botany
University of Colombo
Banana is the most widely consumed fruit in Sri Lanka, and is an attractive perennial fruit crop for small farmers. This is due to its high economic gains throughout the year compared with rice. Lowland rice fields have been converted for banana cultivation. Among the local cultivars, Embul (Mysore, AAB) is in the highest demand for cultivation. Since 1990, the University of Colombo has carried out research on bananas, including micro-propagation through shoot-tip culture, gamma radiation-induced mutations, cell-suspension cultures and somatic embryogenesis, and ploidy analysis for detection of variation.
Since 1995, investigations have been conducted to improve both Embul and Cavendish banana cultivars. Two selections of banana have been made for early fruiting and short height. Micropropagated plants of the selections were tested for trait stability until the second generation. Mass production of plants is in progress. Thus, indexing and testing of plants for viruses, i.e. BBrMV and BSV, has become essential, since virus-free indexed mother stocks are required for micropropagation. Techniques were adopted for routine testing of mother stocks, and random testing of micropropagated plants for BBrMV by DAS-ELISA with imported commercial kits.
The present study was aimed at the development of a low-cost ELISA detection kit. Anti-serum for BBrMV, produced by the Queensland Department of Primary Industries (QDPI), Australia, was tested as the coating antibody to replace the Agdia commercial kit. Results showed a relatively high efficiency with the QDPI antibody. Work is also in progress to make an alkaline phosphatase-conjugated antibody to replace the test kit. With the production of local antiserum, it is expected that an effective low-cost local diagnostic kit could be developed for the routine indexing of banana plants for BBrMV. This would facilitate the identification of virus-free mother stocks for micropropagation. However, purification of the virus extract is still a limiting factor for obtaining the antigen.
Abbreviations: BBTV, Banana Bunchy Top Virus; BSV, Banana Streak Virus; BBrMV, Banana Bract Mosaic Virus; CMV, Cucumber Mosaic Virus; ELISA, Enzyme Linked Immunosorbent Assay; OFC, Other Food Crops; QDPI, Queensland Department of Primary Industries
1.1. Importance of bananas in Sri Lanka
Banana (Musa spp.) is the most widely cultivated and consumed fruit in Sri Lanka. It is also an attractive perennial fruit crop for farmers due to its high economic gains throughout the year. Even rice fields are being converted to grow banana, as it gives more economic benefits, requires relatively less water  and less input than rice, and gives higher returns than rice. The farmer's net profit has gone up about four times compared with rice. In addition, not only is there water saving from banana cultivation, but also the nutritional level in farmers' families has improved due to the habit of increased fruit consumption. Also, there is a reduction in the use of chemical pesticides in banana cultivation, which has a positive impact human on health and the environment. Banana has been given the highest priority amongst Other Food Crops (OFC). Out of the 28 local cultivars, the cultivar Embul (Mysore-AAB) is in the highest demand. Kolikuttu (Silk-AAB) and Ambon (Cavendish-AAA), grown especially in the mid-wet zone of the country, are also in considerable demand.
Currently, nearly 50,000 ha of land is under banana cultivation in Sri Lanka, and the annual banana production is around 450,000 metric tonnes. Until recently, banana cultivation was limited to very small plots, but now large fields are being established. More and more rice farmers are switching to banana cultivation due to the high profit margin and smaller amount of work needed in the field. During the last six years, 2500 ha have been converted to banana cultivation .
Since 1990, under the University of Colombo research programme, research and development work has been carried out on banana micropropagation through shoot-tip culture and mass production of plants. The well developed technology has already been transferred to the farmers [3, 4].
1.2. Gamma irradiation for improved cultivars
Since 1995, under an IAEA/TC program, shoot tips of both Embul and Cavendish cultivars have been gamma irradiated to develop mutant cultivars such as shorter height and earlier fruiting. The irradiation of in vitro shoot tips with 45 Gy resulted in two selections, one with reduced height and the second with earlier fruiting. Both 'Embul' and Cavendish types started to bear fruits after six months from planting . The selected mutants were multiplied by micropropagation, and tested for genetic stability of the newly acquired traits.
1.3. Virus diseases
In Sri Lanka, four major viruses are primarily responsible for poor banana cultivation: BBTV, BBrMV, BSV and CMV. The occurrence of BBrMV and BSV has been reported rather recently. Nevertheless both diseases are spreading widely, especially in the dry zone of the country. A recent survey by the Department of Agriculture  revealed a variable pattern of occurrence of these viral diseases in different cultivars in two main banana-growing areas. For example, the BBrMV infection rate was 2.1 and 1.4%, respectively in cv. Embul and Puwalu, while both cultivars had 3.5 and 1.8% infection rate respectively. Among all the varieties evaluated cv. Embul turned out to be the most highly susceptible cultivar.
Some reports have indicated that BBTV and CMV occur prominently in relation to BBrMV and BSV. The use of infected suckers for the expansion of cultivation was the main reason for the virus spreading. Since micropropagated banana plants are not yet widely used, the chances of virus spreading through this route are negligible. However, this might change in the near future with the use of commercialised tissue-culture technology. Thus, the use of virus-free mother plants in micropropagation becomes very important for the spread of banana viruses.
1.3.1. Banana Bract Mosaic virus (BBrMV)
BBrMV was first reported from the Philippines in 1979  and during 1996 was also found in Sri Lanka . Thomas et al.  confirmed the presence of the causal agent of the disease in the widely cultivated local variety Embul (AAB group, Mysore). In Sri Lanka, the disease is more prevalent in cultivations which are not properly managed, but the impact on the yield is significant.
Since cv. Embul is now being micropropagated, virus-indexed mother plants have become essential. At the same time, the spread of virus diseases in existing traditional small-scale plantations requires monitoring. However, a banana virus indexing kit was not available in the country until recently.
A screening method for the detection of BBrMV has not been adopted (up to 2000) in Sri Lanka, and its diagnosis depends on visible symptoms. Screening should be done with an ELISA test or a PCR-based molecular kit using specific primers. Preferably, an ELISA screening test should rely on locally developed antibodies, since this is more economical for the routine testing of mother plants, for commercial banana micropropagation, and for field cultivation as well.
2.1. Field testing of selected mutants produced by gamma irradiation
Cv. Embul plants, selected for early fruiting, were proliferated in vitro up to M1V8, and 1000 regenerated plants  were transferred to the fields after acclimatization. A dry zone in southern Sri Lanka (Hambantota) and a mid-country wet zone (Ratnapura) were selected for field experiments. Ploidy analyses of plants were carried out using a Flow Cytometer (PARTEC PA II) to detect variation in ploidy level .
2.2. Screening for Bract Mosaic virus (BBrMV)
2.2.1. BBrMV disease symptoms
Disease symptoms of BBrMV were identified in field-grown banana plants at different growth stages. Symptoms on the pseudostem, petioles, leaf sheath and bracts were observed (Figure 1). In the initial stages, symptoms would appear on the leaf petioles and sheath, but when severely affected would appear on the bracts. Samples, mainly from cv. Embul (AAB), were collected for laboratory testing.
2.2.2. Use of Agdia commercial ELISA kit
The commercial ELISA kit, Agdia® (Agdia, USA) was tested, which used DAS-ELISA with polyclonal antibodies (from rabbits) for both the coating antibody and alkaline phosphatase-conjugated antibody. Positive and negative controls were treated according to the manufacturer's instructions. Molecular biology grade chemicals (Sigma) were used as required. Microtitre plates provided with the kit were used and colour development was detected visually, as well as with an ELISA plate reader (BIORAD, 550) at 405 nm.
Several samples with BBrMV symptoms were tested, and different tissues of the same infected plant were also tested. The relationship between the colour development by ELISA and the intensity of disease symptoms was also investigated.
2.2.3. Testing the antibody from antiserum of the QDPI laboratory
Even though the commercial kit gave the expected results, a locally developed kit would be more economical for routine testing. Therefore, a local kit was developed with polyclonal antibodies produced by QDPI, Australia. At the initial stage, this polyclonal antibody serum was used in several dilutions to replace the coating antibody of the commercial Agdia kit, to optimise the dilution and examine the possibility of using the QDPI serum in future.
2.2.4. Sample preparation, extraction and purification of BBrMV
Leaf laminar tissue samples of local banana 'Embul' were collected and stored at 4°C for a maximum of 2 days, for the extraction and purification of BBrMV. All steps of sample preparation were carried out at 5ºC, unless otherwise mentioned. Samples taken from the refrigerator were immediately frozen in liquid nitrogen, and were finely ground by pestle and mortar.
Figure 1 Symptoms of Banana Bract Mosaic Virus (BBrMV) in cv. Embul (AAB). (a) Streaks on bracts; (b) streaks on pseudostem; and (c) split base of sucker.
BBrMV extraction was done according to the procedure given by Thomas et al. . It involved high-speed centrifugation at 10,000 g (HIMAC SCP 85G) and ultracentrifugation at 125,000 g (HIMAC CR 21G). Equilibrium centrifugation in CsCl was done at 126,000 g for further purification. Gradients were collected and concentrations were measured by a UV spectrophotometer (JASCO, Model 7800) at 260 and 280 nm. The fractions with BBrMV were stored at -20ºC for future evaluation of purity, and used to produce antiserum and antibodies.
3. RESULTS AND DISCUSSION
3.1. Field testing of selected mutants induced by gamma irradiation
In the second generation, both early fruiting and short height traits appeared in 80% of Embul (AAB, Mysore type) plants. The other 20% flowered in the seventh or eighth month. With Cavendish, early fruiting was observed in the wet zone under good growing conditions. But in the dry zone and wet zone with poor soils, flowering was observed in the eighth month. Hence it appears that agronomic practices and soil quality have an influence on the selected cultivar. With fertile soil, and agronomic practices recommended by the Department of Agriculture , early fruiting was observed in the selected banana plants. Ploidy analysis of the selected plants did not show any variation in the position of the peaks, indicating no change in ploidy levels of cultivars with improved traits .
Early fruiting and harvesting of micropropagated banana plants saved at least one month compared with conventionally grown planting material, which usually needs 8 months to flower. Thus, the number of ratoons in two years becomes three instead of usual two, thereby increasing the income of farmers by 25%, which is equivalent to about US$ 350 per hectare per year.
3.2. Screening for BBrMV
3.2.1. Disease symptoms
All the currently reported disease symptoms were observed in infected plants in the field, but in varying degrees. Spindle-shaped purple or dark red coloured streak patterns on the pseudostem, in addition to dark spindle-shaped streaks on bracts, were the most common symptoms of infection (Figure 1). The mature plants with inflorescences having black or reddish brown streaks on the outer surface of the open bracts also had streaks on the pseudostem. The splitting of the base of young suckers could also be due to other banana virus infections . Spindle-shaped streak patterns could also occur on the lower side of the petiole of the leaf, and raised veins of the upper surface of the leaf lamina is another symptom. The upper leaf surface may also show chlorotic spindle-shaped yellowish streaks, but it was difficult to relate it to BBrMV infection only as other virus infections may produce similar streaks.
3.2.2. Indexing for BBrMV using the Agdia kit
The Agdia kit gave positive results for all the infected samples. It was noted that the colour intensity of the test results increased with the increase of the severity of disease symptoms. Even in the same plant, some tissues without disease symptoms gave negative results, while parts with symptoms gave positive results. For example, the bracts with streaks gave positive results with the Agdia kit, but flower parts under the same bract were negative. However, for general diagnosis purposes, the commercial Agdia kit could be recommended.
Routine testing of mother stock and random testing of micropropagated plants for the BBrMV by DAS-ELISA is now being carried out. Thus, it is now possible to use BBrMV-free mother stocks for mass production of plants through shoot-tip culture. Development of a local ELISA kit is in progress, and the establishment of the virus-indexing procedure would greatly assure the sustainability of the tissue-culture program, which would also use the mutants developed through mutation breeding.
3.2.3. Use of QDPI antibody
The available polyclonal QDPI antibody at a dilution of 1:400 without further purification was successfully used to replace the original coating antibody of the Agdia commercial kit. The dilution rate of the coating antibody of the commercial kit was 1:200. Therefore, the QDPI polyclonal antibodies would be more economical if other major components of the kit, e.g. alkaline phosphatase-conjugated antibody, could also be prepared from it. The next step would be the preparation of the enzyme conjugate using QDPI antibodies after purification.
3.2.4. Virus isolation and purification
According to Thomas et al. , the virus-containing fraction should give a ratio of 1.17, and therefore, only fraction 3 may contain the virus. However, further evidence is required to confirm that fraction 3 is free from other plant or tissue contaminants. If banana plant contaminants were present, antisera against them would be produced and ELISA would give false positive results. Currently, the virus purification step has become the limiting factor for pure antigen extraction for the antibody production process. The absorption ratio of CsCl extracted fractions is given in Table 1.
Table 1 The absorption ratio of different CsCl extracted fractions
Absorption 260 nm:280 nm
a virus containing fraction according to Thomas et al. .
In Sri Lanka, radiation-induced mutations have been used for banana improvement. A mutant, developed from the cv. Embul (AAB, Mysore type), has been isolated with traits including shorter height and early fruiting. The latter allows the reduction of the fruiting period by one month and thus enables farmers to have four harvests in 24 months from three ratoons. An increase of one ratoon crop has raised annual income of banana growers by about 25%. As a result of the introduction of tissue culture-derived banana plants, the selected mutants were readily multiplied and made available to the growers. There was an increase in demand for micropropagated banana plants, which had a significant impact on the local economy. The most important problem encountered with the production of tissue cultured banana plants during the last few years was the selection of virus-free mother plants.
This project has developed the basic infrastructure at the University of Colombo to develop ELISA diagnostic kits for the detection of BBrMV through technology transfer. The results obtained so far strongly support the potential of developing a local testing kit for detection of BBrMV in Sri Lanka. Experiments are in progress on the extraction and purification of the BBrMV from local banana cultivars.
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