Phytosanitary

One of the goals of in vitro Genebank is to conserve and distribute pathogen-free plant material to be safely exchanged all over the world. Phytosanitary is in charge of the control of diseases in potato and sweetpotato crops guaranteeing plant health.

Currently, 95% and 91% of the in vitro potato and sweetpotato collections are free of principal pathogens (virus, bacterial and fungal), reaching the phytosanitary status “HS2”. The important diseases and their detection methods are well established for potato and sweetpotato, the Andean Root and Tuber Crops (ARTCs) have little information on diseases which infects them. Current research has begun to find efficient methods for diagnosing viruses in ARTCs.

Virus-free accessions of CIP’s in vitro collections
Potato (4,599)95%
95%
Sweetpotato (4,418)91%
91%
Germplasm order form

CIP Genebank accessions available for distribution

Maria Roman
Phytosanitary and Quarentine Specialist

m.roman@cgiar.org

CIP’s virus elimination procedure was originally developed inhouse with help from collaborators and has continuously evolved into an effective but relatively slow, low throughput process that never-the-less can be applied to a wide range of accessions and types of virus. Starting in 2008, methodologies for producing pathogen-tested free materials were improved and certified by the International Standard ISO/IEC 17025:2005.

List of viruses tested by CIP

Potato

Virus Acronym Detection Method
Virus T PVT RT-PCR
Spindle tuber viroid PSTVd RT-PCR
Virus X PVX DAS-ELISA
Virus Y PVY DAS-ELISA
Leaf roll virus PLRV DAS-ELISA
Virus S PVS DAS-ELISA
Andean potato mottle virus APMV DAS-ELISA
Andean potato latent virus APLV DAS-ELISA
Yellowing virus PYV DAS-ELISA
Arracacha virus B- Oca strain AVB-O DAS-ELISA

Sweetpotato

Virus Acronym Detection Method
Feathery mottle virus SPFMV NCM-ELISA
Mild mottle virus SPMMV NCM-ELISA
Caulimo-like virus SPCV NCM-ELISA/PCR
Chlorotic fleck virus SPCFV NCM-ELISA
C6 virus SPC6V NCM-ELISA
Mild speckling virus SPMSV NCM-ELISA
Chlorotic stunt virus SPCSV NCM-ELISA
Latent virus SPLV NCM-ELISA
Cucumber mosaic virus CMV NCM-ELISA
Virus G SPVG NCM-ELISA
Vein clearing virus SPVCV PCR
Begomovirus PCR

RT-PCR/PCR: Reverse transcription – polymerase chain reaction/polymerase chain reaction.

DAS-ELISA: double-antibody sandwich method of the enzyme-linked immunosorbent assay

NCM-ELISA: immuno-enzymatic test that uses nitrocellulose membranes.

The virus elimination process for potato and sweetpotato
Initial health status testing
Health status testing by serological, molecular, and bioindexing (5 months)
Accessions infected with viruses
Accessions found to be infected are subjected to thermotherapy (incubation of in vitro plants at 32-34ºC for potato and 35-37ºC for sweetpotato (~1 month)
Meristem isolation
(0.2-0.35mm meristems)
Plant recovery
Complete plant recovery (2-6 months)
Final health status testing
Step 1 is repeated to confirm that the resulting plants are virus-free ( ~ 5 months).
The virus elimination process for potato and sweetpotato

slide1-phyto

2a.Potato

3a.Potato

4a.Potato

5a2.potato

Meristem growth
Figure 1: Bacteria cleaning
(A) Plants with bacteria (Note red coloration at medium surface (arrow)). (B) Bacteria free plants.

Bacterial contamination in sweetpotato germplasm is about ~ 0.43% (21 accessions) of the collection. The bacterial contamination in sweetpotato appears to be endogenous and involves a suite of bacteria. Infection seems to be connected to the origin of the host plant as virtually all sweetpotato derived from African countries contain some type of bacterial infection. Although it is intriguing to speculate a beneficial effect of such endogenous bacteria in sweetpotato, no evidence of any beneficial effect has been reported. In potato, the incidence of bacterial contamination is less than 0.1% (2 accessions) and therefore is a minor issue.

Bacteria elimination procedures consist of exposing the contaminated accession to a broad-spectrum antibiotic for a controlled time (Figure 1). If the bacteria are not eliminated, the accession is acclimatized in a greenhouse and reintroduced to in vitro conditions with previous disinfection steps.