African Crop Science Journal African Crop Science Society ISSN: 1021-9730 EISSN: 2072-6589 Vol. 5, Num. 4, 1997, pp. 419-424

SHORT COMMUNICATION

Eradication of taro viruses from seedlings via seed rescue culture coupled with thermotherapy

M. E. WAGIH

The Agricultural Biotechnology Centre, Department of Agriculture, The University of Technology, Private Mail Bag, Lae, Papua New Guinea, North Australia

(Received 8 July, 1996; accepted 19 August, 1997)

Code Number:CS97047
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ABSTRACT

In order to investigate appropriate methodologies for producing virus-free taro plants, Papua New Guinea semi-wild taro (SWT), which had been grown in a field germplasm collection of 437 cultivars and exhibited high degree of field resistance to leaf blight and high susceptibility to taro viral diseases, was used as a mother source of seeds. Natural flowering was common, to varying degree, among all cultivars. All SWT plants examined produced 4-5 flower heads per plant with an average of 15,000 potentially viable seeds per head. Seeds were grown aseptically in vitro via a seed rescue culture (SRC) technique and further maintained in an insect-proof greenhouse for a period of 8 months and regularly inspected. Plants regenerated from seeds treated at 60 C for 120 min and at 55 C for 120 min remained free of all taro viral disease symptoms. Both treatments, however, significantly reduced germination from 92.16% to 42.24% and 62.28% respectively. Six other treatments, although all reduced symptoms below those of the untreated control, failed to various degrees to eradicate the symptoms. In addition to morphological and physiological variability among seedlings, the plants showed a range of susceptibility to leaf blight. The importance of using thermotherapy with the SRC technique in taro germplasm conservation, breeding and quarantine is highlighted.

Key Words: Alomae and bobone viruses, Colocasia esculenta, dasheen mosaic, tissue culture, thermotherapy

Resume

Afin qu'on puisse mener a terme les methodologies appropriees pour cultiver les choux caraibes, les choux caraibes a croissance demi-spontanee de Papua New Guinea (swt), dont la variete avait ete cultivee sur un champ de collection du plasme germinatifet ayant aussi 437 varietes et dont la resistance est tres elevee a la brulure des feuilles et aussi la sensibilite egalement elevee aux maladies virales, la variete etait utilisee comme source principale de multiplication de semences. La floraison naturelle etait habituelle, mais aux degre variants, parmi les varietes. Toutes les varietes examinees on produit 4 a 5 boutons a fleurs par plante avec une moyenne de 15,000 graines, d'une potentialite viable par bouton a fleurs . Les semences ont ete cultivees aseptiquement in vitro en utilisant une technique de conservation de semences, qui ont ete ensuite, conservees dans les serres anti-parasitaires pour une periode de 8 mois et sous la surveillance reguliere. Les plantes regenerees des semences traitees a 60 C et 55 C respectivemment pour une duree de 120 min ont demeure saines des symptomes des maladies virales de chou caraibe. Les deux traitements, par contre, ont reduit, d'une maniere considerable, leur faculte germinative de 92.16% a 42.24% et 62.28% respectivemment. Malgre quils aient pu reduire les symptomes des maladies aux niveaux plus bas que les echantillons moins non traites, les experiences menees sur six autres traitements, n'ont pas reussi a eliminer, aux degres variants, les symptomes des maladies. En plus de la variabilite morphologique et physiologique parmi les semis, les plantes ont demontre une sensibilite variante a la maladie des brulures des feulles. L'importance d'utiliser la thermotherapie avec la technique (SRC) dans la conservation du plasmegerminatif, la selection et l'etablissement des qarantaines est mis au point.

Mots Cles: Alomae and bobone viruses, Colocasia esculenta, dasheen mosaic, tissue culture, thermotherapy

INTRODUCTION

Diseases of taro, Colocasia esculenta ((L.) Schott), caused by fungi, bacteria, viruses or other pathogens, result not only in subsequent reduction in vigour, quality and yield of the crop, but also constitute a barrier to international exchange of its germplasm. Unlike fungal and bacterial diseases of taro, viral diseases are not curable with any commercially available treatment.

Three taro viruses with different particle morphology have been reported to be present in many countries including Papua New Guinea (Jackson, 1978; Brunt et al., 1990). Two viruses namely dasheen bobone rhabdo virus (DBRV) and dasheen badna virus (DBV) are both bacilliform in shape with particle size of about 330nm x 50 nm and 125 nm x 30 nm, respectively. The third virus known as dasheen mosaic potyvirus (DMPV), has filament flexous rods, 750 nm long (Brunt et al., 1990). Despite the agronomic significance of taro viruses, their economic impact and contribution to the decline of the crop has not been thoroughly investigated. As taro is vegetatively propagated, the viruses disseminate with propagation of infected planting material through successive generations, which can result in virtually the entire crop becoming infected.

Currently, there is restriction on the transfer of taro germplasm from disease-infected areas to other disease-free areas where they can be used for planting, germplasm collections or in breeding programmes. Due to the lack of convenient and reliable diagnostic techniques for detecting taro viruses, particularly alomae and bobone, and of techniques aimed at elimination of those viruses, cultivars are presently quarantined conventionally in order to avoid accidental introduction of the diseases. This procedure is labour intensive, time consuming, expensive, and requires considerable investment in glasshouse space.

Tissue culture has assumed considerable importance as a method of producing disease-free taro plants. In general fungal and bacterial pathogens as contaminants are routinely eradicated. Viruses, however, are much more difficult to eliminate. In vitro meristem-tip culture technique alone, or with the aid of thermal or chemical therapy or a combination of both, has been frequently used to eliminate systemic viruses from various crop plants (Wagih et al., 1995). Meristem-tip culture of taro has commonly been used for the production of virus-free clones (Hartman, 1974; Jackson et al., 1977; Arditti and Strauss, 1979; Strauss and Arditti, 1980). However, this technique may be confounded by low regeneration rates, failure of some regenerants to be virus-free and smaller meristems resulting in lower numbers of virus-free plants. Additionally, callus occurrence in meristem-tip cultures leads to regeneration of undesirable off-types (Kartha, 1981). Hence, vigorous virus-testing and quarantine procedures are still deemed necessary.

Virus-testing in taro is of extreme importance. FAO/IBPGR (Zettler et al., 1989) have provided guidelines for the exchange of aroids stating that seeds of Colocasia and other aroids pose a minimal risk of introducing exotic pathogens. If it is not essential to move particular genotypes of aroids, seeds should be preferred for the movement of its germplasm (Volin and Zettler, 1976; Strauss et al., 1979). They recommended a post-entry quarantine period of one crop cycle.

A seed rescue culture technique, described by Wagih (1994) offers a means for mass propagation of taro under in vitro conditions for protection from reinfection by taro viruses. The present study describes the establishment of metho-dologies for mass propagation of virus-free taro seedlings via seed rescue culture coupled with thermotherapy, in order to minimise the risk of accidental introduction of taro pathogens to 'clean' areas and to facilitate exchange of pathogen-free germplasm between taro genebanks and users.

MATERIALS AND METHODS

Source of seeds. Papua New Guinea semi-wild taro showing a high degree of field horizontal resistance to leaf blight (Phytophthora sp.) was used as a mother source of seeds. Plants were intercropped with 437 local cultivars in a germplasm collection maintained at Bubia Agricultural Research Centre, near Lae. Plant material showed severe symptoms of taro viral diseases, specifically dasheen mosaic and the alomae/babone complex.

Extraction and surface sterilisation of seeds. Seeds were extracted from ripe fruit heads by macerating the fruits and washing the seeds on a piece of cloth (0.8 x 0.8 mm mesh) placed over a beaker filled with water. Seeds remained on the screen while the fruit pulp dissolved in water passed through the screen. Remaining seeds were thoroughly washed in water and freed from any debris. Seeds were surface sterilised by rinsing in 1.5% chlorine (sodium hypochlorite) containing Tween 20 (one drop per 100 ml solution) for 15 min, and then washed three times in sterile distilled water.

Thermal treatment of seeds. Seeds were thermally treated in sterilised ELISA microplate wells (12 x 8 = 98). Under aseptic conditions, microplates were charged by placing a single sterilised seed into each well containing 2ml of half-strength liquid MS medium (Murashige and Skoog, 1962). Charged plates were then wrapped with parafilm and placed in a plastic container in a Thermomix water bath set at a treatment tempe-rature and covered with a glass lid. Only one plate was heat-treated at any one time.

Culture method. Seed rescue cultures were initiated as described by Wagih (1994) using a basic semi-solid MS medium, modified by the addition of NAA (0.5 gL^-1), BA (0.5 gL-l) and taro extract (20 ml L^-1). Cultures were incubated at 28 + 2 C day and 20 + 2 C night with a photoperiod of 18 hr per day (light intensity 150 E m^-2 s^-1).

Regenerated plantlets with shoots about 8-12 cm long and substantial root were transferred to autoclaved "Jiffy" ready pots and enclosed in transparent plastic bags supported above by wire struts. The pots were maintained in a greenhouse and watered weekly with sterile tap water through a small opening in the plastic bag, until they reached a height of 15-20 cm. Plants were then transferred to 20 cm diameter pots containing sterile top soil, placed in an insect-free greenhouse and watered on alternate days. Plants were regularly and critically inspected for development of taro disease symptoms over an eight month period. Taro leaf blight, dasheen mosaic, and alomae/babone diseases were diagnosed by their characteristic symptoms as described by the South Pacific Commission (Jackson, 1977, 1978; Zettler and Jackson, 1979).

RESULTS AND DISCUSSION

Regeneration of taro from seeds. Taro seed populations are highly polymorphic and produce highly heterogeneous seedlings. This degree of diversity suggests a high degree of heterozygosity due to cross-pollination which is believed to be common in taro (Lebot and Aradhya, 1992; Wagih, 1994). Taro seeds grow well in vitro and in soil. Wagih (1994) described a seed rescue culture technique for the regeneration of contaminant-free in vitro seedlings at a regeneration rate of 96%. In that study, seeds failed to germinate in autoclaved dampened topsoil, in the field or under high relative humidity. However, in the current study over 1000 seeds germinated and grew easily in soil, essentially using a technique described by Dr. A. Ivancic, Bubia Agriculture Research Centre, Lae (pers. comm, 1993), by broadcasting on sterilised topsoil in 30 cm diameter pots dipped in a 10 cm deep water bed. Germination occurred over a period of 7-14 days and seedlings with 2-3 leaves were singled out and maintained.

Eradication of viruses from seeds. Taro viruses are believed not to be seedborne but this has yet to be confirmed. The uncertainity in using taro seeds (and seeds from other aroids) as a source of virus-free seedlings was expressed in a recommendation by FAO/IBPGR (Zettler et al., 1989) which emphasised that taro seed should be germinated and the resultant seedlings grown in quarantine for one crop cycle before release.

In an attempt to answer the question of whether taro seeds are a source of virus-free seedlings and to study the effect of heat on eradicating taro viruses from seeds (if present), seeds were exposed to heat treatments after surface sterilisation and prior to tissue culture.

Table 1 shows the effect of a range of heat treatments on the condition of the seed rescue cultures and the occurrence of symptoms typically caused by taro pathogens. In this experiment, seeds were extracted from taro plants showing severe symptoms of taro leaf blight, dasheen mosaic, and the alomae/bobone complex. They were tissue cultured and maintained in an insect-proof greenhouse to prevent reinfection. Under the assumption that taro viruses are not seed- borne, plants from contaminant-free cultures which were maintained under insect-proof conditions were expected to remain free of taro viral disease symptoms. Consequently, the occurrence of symptoms in control plants suggests the pre-existence of the causal viral pathogens in the taro seeds. This finding may justify the suspicion expressed by FAO/IBPGR (Zettler et al., 1989) that seeds of taro still pose a risk of introducing taro viruses into new areas. It has been argued that symptomless plants may not be viruse-free as latent viruses may still be present (Jackson, 1978). Therefore, a period of one crop cycle (7-8 months) is believed to be adequate to allow for the multiplication of virus particles, if present, and reappearance of symptoms on seedlings following regular and critical visual inspection over an eight months period suggests successful eradication of the viruses (Zettler, pers. comm. 1993).

Out of the eight thermal treatments applied to seeds prior to culturing, only two were found to eradicate all taro viruses. These two treatments were 60 C for 120 min and 55 C for 120 min which allowed the recovery of 42.24% and 62.28% of the seedlings, respectively. Out of 25 plants maintained from each of the two treatments, none developed any symptoms of taro viral diseases over the eight month period. Since the emphasis of this work was production of virus-free taro, these two treatments are considered valuable. The latter treatment, however, is highly recommended as the germination rate after seed treatment was significantly (P < 0.05) higher than the other treatment. On the contrary, the remaining six thermal treatments failed, in varying degrees, to eradicate the alomae/babone virus complex. However, seed treatment at 55 C for 60 min, 60 C for 30 min, and 60 C for 60 min resulted in the eradication of dasheen mosaic virus only. This result shows that dasheen mosaic virus is more quickly eradicated by, and therefore perhaps more sensitive to, high temperature than the alomae/bobone virus complex.

Despite the fact that the mother plants used as a source of seeds, had shown a high degree of resis-tance to Phytophthora, leaf blight symptoms app-eared on all seedlings. This may suggest that the leaf blight resistance may be due to anatomical features of the field grown plants which may have been abscent in seedlings grown in vitro and under greenhouse conditions.

The successfully regeneration of virus-free seedlings of taro in this study should significantly improve the safe movement and enhancement of taro germplasm. Seeds and seedlings could also substitute for lost cultivars by recovery of elite genes which could be useful in conventional and, to a larger extent, biotechnological breeding programmes. If homozygous elite taro cultivars are achieved, resultant seeds may be used for micropropagation of taro clones at a regeneration rate of up to 60,000 seedlings/plant or plantlets regenerated from seeds could be conventionally propagated. These potential uses of taro seeds and seedlings should facilitate the exchange of its germplasm and passage through quarantine and enhance international collaboration on taro breeding and improvement.

ACKNOWLEDGMENT

I am grateful to Dr K.S. Powell and the late Dr. J. C. Reid for comments and critical review of this manuscript.

REFERENCES

Arditti, J. and Strauss, M.S. 1979. Tissue culture propagation of taro (Colocasia) and Xantho-soma. Information Document 44. South Pacific Commission, Noumea, New Caledonia, 57 pp.

Brunt, A., Crabtree, K. and Gibbs, A. 1990. Viruses of Tropical Plants. CAB Internation. Redwood Press Ltd. 707 pp.

Hartman, R.D. 1974. Dasheen mosaic virus and other phytopathogens eliminated from caladium, taro and cocoyam by culture of shoot tips. Phytopathology 64:237-240.

Jackson, G.V.H. 1977. Taro leaf blight. Advisory leaflet No. 3, South Pacific Commission, Noumea, New Caledonia.

Jackson, G.V.H. 1978. Alomae and bobone diseases of taro. Advisory leaflet No. 8, South Pacific Commission, Noumea, New Caledonia.

Jackson, G.V., Ball, E.A. and Arditti, J. 1977. Research note: Seed germination and seedling proliferation of taro, Colocasia esculenta (L.) Schott in vitro. Journal of Horticultural Science 52:169-171.

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Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Planta-rum 15:473-491.

Strauss, M.S. and Arditti, J. 1980. Plantlet regeneration from shoot tip cultures of Xanthosoma caracu. Annals of Botany 45: 209-212.

Strauss, M.S., Michaud, J.D. and Arditti, J. 1979. Seed storage and germination and seedling proliferation in taro, Colocasia esculenta (L.) Schott. Annals of Botany 43:603-612.

Volin, R.B. and Zettler, F.W. 1976. Seed propagation of cocoyam, Xanthosoma caracu Koch and Bouche. Horticultural Science 11: 495-460.

Wagih, M.E. 1994. Seed rescue culture: A tech-nique for the regeneration of taro (Colocasia esculenta). African Crop Science Journal 2: 9-15.

Wagih, M.E., Gordon, G.H., Ryan, C.C. and Adkins, S.W. 1995. Development of an axillary bud culture technique for Fiji disease virus elimination in sugarcane. Australian Journal of Botany 43:135-143.

Zettler, F.W. and Jackson G.V.H. 1979. Dasheen mosaic virus. Advisory leaflet No 10, South Pacific Commission, Noumea, New Cale-donia.

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Copyright 1997, African Crop Science Society

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