3.7.8
PROPERTIES AND INCIDENCE OF A PREVIOUSLY UNREPORTED SEEDBORNE FILAMENTOUS VIRUS INFECTING CITRUS IN NEW ZEALAND

MN PEARSON1, M AFTAB1 and P MOONEY2

1School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand; 2The Horticultural and Food research Institute of New Zealand, Kerikeri Research Centre, PO Box 23, Kerikeri, New Zealand

Background and objectives
Soon after the establishment of Satsuma orchards in the mid 1980s virus-like disease symptoms were observed. The symptoms included boat and spoon shaped leaves, dwarfing of plants and small fruit size. Research was initiated to characterise the virus(es) associated with the disease symptoms.

Materials and methods
Viruses were purified from homogenised leaf and/or bark material by two cycles of precipitation with ammonium sulphate and PEG followed by one or more cycles of differential ultra-centrifugation and density gradient centrifugation in cesium sulphate. Sap extracts and purified virus were tested against antisera to satsuma dwarf virus (SDV), citrus tristeza virus (CTV), citrus tatter leaf (CTLV) and and citrus ringspot (CRSV) (Indian isolate), by ISEM and ELISA. The capsid proteins of purified virus particles were separated by SDS-PAGE and amino acid sequence obtained by HPLC.

Results and conclusions
Sap extracts from diseased satsumas were mechanically inoculated to 20 different herbaceous plant species, including Phaseolus vulgaris, Chenopodium quinoa and Vigna unguiculata, which are known indicators for CTLV and CRSV. Phaseolus vulgaris sometimes exhibited systemic pinpoint chlorotic spots; none of the other species expressed any symptoms. Electron microscopy of purified preparations showed the presence of two distinctly different types of filamentous particles. One was identified as CTV, based on morphology and ELISA. Particles of the second virus were of variable in length, probably due to fragmentation [1]. Negatively stained particles from leaf dips mostly ranged in length from 150-500 nm. The virus was subsequently found in a range of other citrus species, most plants were co-infected with CTV and a range of symptoms were expressed. It was also found in symptomless, CTV-free, seedlings of Madame Vinous and C. excelsa, grown in an insect-free glasshouse, and was provisionally named citrus seed borne virus (CSBV). CSBV did not react with antisera to SDV, CTV or CTLV but reacted strongly, both in ELISA and ISEM, to antisera raised against an Indian citrus virus recorded as citrus ringspot [2]. However, CRSV is not known to be seed borne and neither the Indian or NZ viruses induced local lesions on Chenopodium quinoa. In addition PCR products were not obtained from CSBV using primers specific to a Florida citrus psorosis-ringspot isolate (Barthe, pers. comm.) and the particles of CSBV do not have the spirovirus morphology typical of Florida CRSV isolates. When analysed by SDS-PAGE samples containing only CSBV produced two protein bands of 33 and 25 kDa (CTV produced a band of 28 kDa). Amino acid sequence has been obtained from the 33 and 25 kDa bands and primers have been designed to amplify part of the CP gene by PCR.
It is concluded that citrus in New Zealand is commonly infected by a seed borne filamentous virus. The virus is not related to CTV, CTLV or US strains of CRSV, but is serologically related to an Indian virus isolate also referred to as CRSV.

References
1. Jagiello C, Mooney P. Dawson,E, Aftab M, Pearson M, 1995.The Orchardist, Sept 1955 pp. 40-44.
2. Byadgi AS, Ashlawat YS, Chakraborty NK, Varma A, Srivastava M, Milne RG, 1993. Proceedings 12th Conference of the International Organization of Citrus Virologists, pp. 155-162.