ABSTRACT

Leaf and stem tissue from nine citrus cultivars collected from the Lower Rio Grande Valley and Corpus Christi areas of Texas were biologically indexed for citrus tatterleaf (CTLV). Sixteen of the 25 samples induced CTLV - like symptoms in citrange or citrumelo seedlings. Leaflet symptoms included; notching, distortion, vein clearing and asymmetry associated with chlorosis. Mechanical inoculations with three isolates on cowpea and red kidney bean, produced variable symptoms ranging from none to leaf chlorosis, discrete local lesions, systemic vein necrosis, and shoot epinasty. Antisera to CTLV (Japan) and apple stem grooving virus (ASGV; UK and Germany) were used in enzyme-linked immunosorbent assays (ELISA). The CTLV and ASGV (Germany) antisera detected the antigen in seven citrus samples and three ASGV standards. The ASGV (UK) antisera detected two of the CTLV isolates.

INTRODUCTION

Importance of CTLV to Texas. The majority of citrus in Texas is grown on sour orange rootstock which is sensitive to citrus tristeza virus (CTV). The recent movement of the efficient CTV vector, the brown citrus aphid, through Central America, the Caribbean and Florida may prompt many growers to use CTV tolerant rootstocks such as the trifoliate orange, (Poncirus trifoliata (L) Raf.), citrange (P. trifoliata x Citrus sinensis L.) And citrumelo (P. trifoliata x C. paradisi Macf.). These rootstocks are sensitive to CTLV. Meyer lemon (C. Meyeri Y. Tan.) Was imported from China and grown commercially in the Lower Rio Grande Valley of Texas early this century. Propagations of the initial imports were found to harbor CTLV.

CTLV is transmitted with ease by mechanical means and is difficult to remove by shoot-tip grafting thermotherapy treatments. A reliable method to detect CTLV is essential for the Texas budwood certification program.

CTLV symptoms. A bud union necrosis or abnormality can develop when symptomless CTLV carrying citrus, such as sweet orange, sour orange, grapefruit (C. Paradisi Macf.), mandarin (C. Reticulata Blanco) or lemon (C. Limon (l.) Burm.f.) Is budded onto a trifoliate orange or a trifoliate hybrid rootstock. Deep fluting of the rootstock trunk and an extended bud union crease or gap can result as early as eighteen months after grafting (Fig. 1). In high winds, the scions may sever completely at the bud union.

The objectives. To partially characterize CTLV isolates from Texas using citrus and herbaceous indicators and to test available antisera for rapid diagnosis.

METHODS

Indexing onto citrus plants

Donor tissue grafted to recipient Rusk, Carrizo, Troyer citrange or Swingle citrumelo.                        

Indexing onto herbaceous plants

Sap inoculations onto cowpea (Vigna unguiculata (L.) Wolp.) And red kidney bean (Phaseolus vulgaris L.).

Serology

Antisera used: CTLV-J (Japan), ASGV-L (Germany), ASGV-A (UK). In double antibody sandwich ELISA.

CTLV-J antisera were kindly donated by Dr. A. Kawai, Japan. ASGV-L antisera and controls were purchased from Loewe Biochemica, Germany (ASGV-L). Dr. A. Adams, UK, donated crude ASGV antiserum, which was purified and conjugated during this study (ASGV-A). Dr. G.I. Mink, USA, donated in planta ASGV isolates.

RESULTS AND DISCUSSION

Symptoms of CTLV in indicator citrus. CTLV-like symptoms were present in sixteen out of the 25 samples tested and the TL-100 CTLV control isolate.

Rusk, Carrizo and Troyer citrange showed asymmetry and notching of young leaflets. Under a bright background light, the notches were found associated with chlorotic patches as early as 28 days after inoculation. Leaflet distortion or buckling was later observed in the same leaflets, and in new leaflets in the same branch. Chlorotic spots, of variable diameter were observed in the leaflets of inoculated plants (Figs. 2 & 3). Symptoms were more distinct in Rusk citrange than Troyer or Carrizo.

Swingle citrumelo inoculated with TL-100 tissue had a pronounced stem epinasty or zig-zag type growth. Leaflet asymmetry and deformity occurred with distinct vein clearing and associated chlorotic patches (Fig.4).

Stunting of some of the inoculated plants was also noted (CTLV symptoms have been referred to as citrus tatterleaf and citrange stunt by previous workers).

Symptoms of CTLV in herbaceous indicators. Symptoms in inoculated red kidney bean plants were variable depending on the CTLV isolate, ranging from no symptoms, a vein necrosis in primary leaves to dark brown local lesions. Local lesions were not consistent in inoculated cowpea (Table 1).

 ELISA. CTLV-J antisera were compared to ASGV-L and ASGV-A antisera in parallel assays. The CTLV-J and ASGV-L antisera reacted with seven CTLV isolates, and detected ASGV positive controls from the USA and Germany. ASGV-A detected to of the CTLV isolates detected by CTLV-J and ASGV-L and all ASGV positive controls. Most detectable samples derived from tissues of CTLV symptomless citrus; Meyer lemon, Etrog citron (C. Medica L.) Or Rough lemon (C. Jambhiri Lush.). The trifoliate hybrids used in the indexing may be sensitiva to relatively small amounts of virus for symptom expression, whilst the CTLV symptomless citrus may provide higher titres of the virus. Indexing based on ELISA is relatively rapid, efficient, sensitive to the virus, and can provide more evidence as to the CTLV status of different samples.

ACKNOWLEDGEMENTS

Financial support for this project was from the United States Department of Agriculture - Agency for Plant Health Inspection Service - Plant Protection and Quarantine (USDA-APHIS-PPQ) through a Co-operative Agricultural Pest Survey (CAPS) agreement to Dr. Mani Skaria and Dr. J. Victor French. Thanks are also due to Dr. Richard F. Lee, University of Florida, Citrus Research and Education Center, Dr. Robin G.T. Hicks, School of Biology and Biochemistry, University of Bath, UK, and the University of Bristol, UK, for support in various aspects of this work.

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