Partial Characterisation of a Filamentous Seedborne Virus Infecting New Zealand Citrus

by Aftab, Mohammad

A short filamentous virus (SFV), initially isolated from Satsuma mandarin, was found in a wide range of citrus species. The virus was in low concentration in citrus tissue and was not detected by transmission electron microscopy of negatively stained crude sap extracts. The virus was difficult to purify, but by using a combination of sequential virus precipitation with ammonium sulphate and polyethylene glycol, followed by differential centrifugation and caesium sulphate gradients, adequate amounts of virus could be obtained for antiserum production and protein analysis. Several antisera raised in rabbits were evaluated using ISEM and ACP-ELISA. ISEM was found to be the most suitable method for detecting SFV in crude sap extracts. Virus particles from purified preparations and trapped from the crude sap by ISEM were of consistent appearance and diameter, but of variable length. One hundred particles trapped from crude sap extracts of the seedlings of Madam Vinous and Citrus excelsa using SFV-specific antiserum, ranged in length from 146 to 948 nm. While 85% of the particles were between 150 to 500 nm, there was no clear modal length. Similar measurements were obtained from SFV purified from field trees of satsumas, Dweet tangor and Thorny mandarin.

SFV was seed transmitted in 17 from 19 seedlings species tested; only the seedlings of two rootstocks, Poncirus trifoliata cv. Benecke and Zhuluan, tested virus-free by ISEM. None of 25 field grown cultivars tested were found to be virus-free. The virus was symptomless in all of the seedling. The only symptoms noted in field plants were boat and spoon shaped leaves in satsumas and leaf flecking in Dweet tangor. Since most field grown citrus in New Zealand is contaminated with CTF it is not certain that SFV was responsible for these symptoms. With phosphate buffer extraction (pH 7) the virus was not mechanically transmitted to any 12 herbaceous hosts tested, belonging to 4 different families. On the basis of the evidence to date the host range of SFV appears limited to citrus.

The physical, biological and serological properties of SFV were compared with those of previously reported filamentous viruses of citrus, but there was no complete correlation with the properties of CTV, CTLV or CRSV (psorosis). Morphologically the SFV particles are similar to the L particles of CRSV (Indian isolates). There is a clear difference in morphology between SFV particles and those of CTV and CRSV (Florida isolate and California isolate). CTLV and CRSV (California and Florida isolates) are mechanically transmissible to several herbaceous hosts, while CRSV (Indian isolate) and SFV are not. SFV is seed transmitted at a high rate while none of the other filamentous citrus viruses are reported to be seed transmissible, except for CRSV where conflicting reports exist. SFV did react with an antiserum to CRSV (Indian isolate) although both the size and number of capsid proteins are different. It did not react with antisera to any of the other filamentous citrus viruses.

Samples of purified SFV gave two prominent protein bands in SDS-PAGE, with molecule weights of 33 kDa (upper hand) and 24 kDa (lower band). Partial amino acid sequence was obtained from these two protein species. On the basis of N-terminal sequence and portions of the internal sequence SFV-specific primers were designed and used in PCR. Nucleic acid extraction from purified virus produced a single high molecular weight band of approximately 11.5 kb in agarose gels. The purified RNA was used as a template for cDNA synthesis by reverse transcription followed by PCR which yielded two bands (700 and 200 bp) amplification products.

It is concluded that the properties of the short filamentous virus isolated from citrus in New Zealand did not consistently match those of any of the previously described flexous viruses from citrus. On the basis of the characteristics of SFV the name” citrus seedborne virus CSBV)” is suggested.