Response of several genotypes of citrus and citrus relatives to 
viroid infection

SUMMARY

Species of citrus and related genera belong to the family Rutaceae. Citrus are 
natural hosts of seven viroids, but only Citrus exocortis viroid (CEVd) and specific 
variants of Hop stunt viroid (HSVd) cause diseases (exocortis and cachexia, 
respectively) in sensitive species. Most citrus are tolerant to viroid infection except 
some species that are sensitive and develop symptoms. In infected commercial 
cultivars, viroids are usually found as multiple infections. The aim of this work is to 
study the response of several citrus genotypes to viroid infection.
A field isolate was characterized and shown to contain CEVd (class A), HSVd 
(non pathogenic variant), CBLVd (CVd-Ia variant) and CDVd (CVd-IIIa and CVd-IIIb 
variants). The effect of this isolate on the performance of 'Nules' clementine and 
'Navelina' sweet orange trees grafted on Carrizo citrange and maintained during a 10 
years in the field was studied. Infection resulted in small trees yielding a crop smaller 
than that of the non-infected controls. Fruit characteristics were also affected: (i) Sweet 
orange fruits from infected trees had size, maturity indexes and juice content higher 
than those from non-infected trees; (ii) clementine fruits from infected trees were larger 
and presented a diameter/height ratio larger than those from non-infected controls; (iii) 
in both species, juice density, amount of soluble solids and acidity of fruits from 
infected trees were lower than those of fruits from non-infected controls. Infected trees 
also had a poorly developed root system with fibrous roots containing fewer 
amyloplasts than non-infected trees. The effect of viroid infection on the root system 
was confirmed through an in vitro assay that showed that explants from infected 
Carrizo citrange had lower rizhogenesis capacity that those of non-infected ones.  
Preliminary studies showed that Citrus karna was susceptible to viroid infection 
and that the inoculated viroids reached high concentrations in the infected plants. By 
comparing the distribution of viroids in C. karna and Etrog citron it was found that both 
hosts contained high viroid concentrations in the bark of young shoots which confirmed 
not only the infection but their long-distance movement through the phloem. However, 
analysis of leaves showed that the viroid titters in C. karna were very low, and 
sometimes even imperceptible, whereas in Etrog citron they were much higher. Viroids 
were undetectable in preparations of mesophyll protoplasts from C. karna leaves while 
in Etrog citron they were found at high concentrations, indicating that unlike Etrog 
citron, viroids are phloem restricted in C. karna.
In studies addressed to identify genotypes resistant to infection with citrus viroids, 
Eremocitrus glauca and Microcitrus australis, two species of citrus related genera were 
selected because their evolution in an unusual habitat. Infection, movement and 
accumulation of CEVd, HSVd, CBLVd, CDVd, CBCVd and CVD-V were studied in self-
rooted plants, plants grafted on rough lemon and plants top grafted with Etrog citron. 
The plants were analyzed by northern hybridization and RT-PCR. The results indicated 
that M. australis, and in particular E. glauca, are poor viroid hosts, but that both 
genotypes allowed the long-distance movement of viroids to the rootstock as well as to 
the top-worked Etrog citron. 
Different isolates recovered from commercial cultivars of three different citrus 
industries have been characterized. In Sudan and Iran, two countries in which 
sanitation and certification programs have not been developed, viroids were found to 
be widespread as multiple infections. Since in both citricultures the main rootstock is 
sour orange and Citrus tristeza virus (CTV) has been detected, the presence of viroids 
should be taken into account when designing strategies to control the damage 
produced by CTV. Cloning and sequencing of HSVd variants from Sudan led to the 
identification of sequence variants in which the composition of the cachexia expresion 
motif differed from the variants described earlier. In Spain, despite the implementation 
of citrus Sanitation and Certification Program, orchards established with viroid-infected 
trees grafted on Carrizo citrange can still be found in certain areas in which trees 
present a poor performance that affects tree size and fruit production.