Seed health testing and pest detection is a first-line approach in managing seed-borne and seed-transmitted pests. In the case, of true seed crops, some pests infecting host crops are seed-borne (e.g., Fusarium oxysporum
in cowpea), some are seed-transmitted (e.g., bean common mosaic virus in cowpea and common bean), and some are either seed-borne or seed-transmitted (e.g., Phyllachora maydis,
which is responsible for tar spot affecting maize) [10
]. Seed-borne and seed-transmitted pests are a concern for germplasm conservation and exchange, and procedures are therefore used to eliminate pests including the use of seed treatment methods or the regeneration and harvesting of seed from healthy plants. However, most pests affecting vegetatively propagated crops, especially intracellular pests, such as phytoplasmas, viruses, and viroids, can spread through vegetative propagules, and eliminating them requires the use of complex procedures. The GHUs routinely check for about 320 pests that are endemic in germplasm production sites, including bacteria, fungi, insects, nematodes, oomycetes, phytoplasmas, viruses, and viroids (Supplementary Table S2
). The testing also covers other pests listed in the import permit of the country that receives germplasm. According to the crop mandate of the center, each GHU is specialized in enabling the production of quality germplasm in accordance with the best procedures available for the diagnosis and detection of pests, treatment for phytosanitation, and international transfers. GHUs apply similar procedures for genebanks and breeding programs, although genebank materials are more diverse, including wild species, landraces, and new acquisitions from new collection missions, which may demand complex/time-consuming procedures, owing to the different species biology and pest risks. The breeding program materials mostly comprise staple cereals, grain and oil seed legumes, roots, tubers, and banana crops. In general, managing the phytosanitary risks associated with true seed crops is relatively easy and effective, as not all the pathogens and pests are seed-transmitted, or seed-borne. Moreover, it is relatively easy to control or eliminate infections of seed-transmitted, and seed-borne pests from seed using chemical or heat treatments, thus salvaging germplasm. In the case of clonally propagated crops, however, systemically infectious pathogens, especially viruses and viroids, are difficult to eliminate without applying complex procedures, which are expensive and time-consuming. Brief details on the procedures employed to generate pest-free germplasm by crop group are summarized here.
4.1. True Seed Crops
Cereal germplasm from breeding programs and genebanks is inspected for both seed-borne and seed-transmitted pests. General procedures for testing, detection, diagnosis, and seed treatment for the elimination of seed-borne pests are used [51
], including the International Seed Trade Association (ISTA) methods, where applicable [53
]. The general phytosanitary procedures used for true seed phytosanitation include, (i) active-growth stage inspection at the flowering/pre-harvest stage to check for the presence of any regulated pests and seed-transmitted pests; (ii) dry seed examination using a desk magnifier (2x) to remove the admixtures of plant debris, sclerotia, galls, insects, smut sori, and discolored and moldy seeds; (iii) seed-washing and a sedimentation test to detect the spores that could not be detected either in dry seed examination or incubation tests; (iv) standard blotter tests to detect the presence of fungi; (v) an agar test (selective media) to detect the bacterial pathogens using specific media; (vi) a seed soaking test to detect the presence of nematodes; (vii) a seed treatment involving a fungicidal treatment to remove saprophytic fungi and seed-borne pathogens; and (viii) seed fumigation using aluminum phosphide (or methyl bromide for sorghum seeds, as per the requirement of Indian NPPO at ICRISAT, India) [54
]. The tests performed for some important seed-borne and seed-transmitted diseases of various CGIAR mandate crops are summarized below.
Barley: The most important seed-borne fungi are smut (Ustilago nuda), covered smut (Ustilago hordei), spot blotch (Bipolaris sorokiniana), head blight (Fusarium graminearum), barley leaf stripe (Pyrenophora tritici-repentis), ergot (Claviceps purpurea), a bacterium responsible for basal glum rot (Pseudomonas syringae pv. atrofaciens); a virus (barley stripe mosaic virus (BSMV)), a seed gall nematode (Anguina tritici), and an insect, the khapra beetle (Trogoderma granarium). Standard phytosanitary procedures are used to test and generate pest-free germplasm for import and export, including considerations of the additional conditions laid down by the NPPO of the import and export countries.
Maize: The main risks associated with maize germplasm exportation are associated with pathogens, such as Pantoea stewartii pv. stewartii maize dwarf mosaic virus, maize chlorotic mottle virus, sugarcane mosaic virus, and wheat streak mosaic virus, which have a restricted geographical distribution. These pathogens are proven to be seed-borne and seed-transmitted, although some of them have a low transmission rate of <1%. Many other maize pathogens are listed in the requirements of the country importing the germplasm, and the measures taken to guarantee that seeds are pathogen-free cover a wide range of possible threats by applying strict phytosanitary procedures in the multiplication field plots and exhaustive laboratory seed testing using conventional, serological, and molecular methods and seed treatments.
Rice: Many pests and pathogens have been identified as posing a risk to rice germplasm. GHUs use various procedures, as summarized above, for seed-borne pests, including bacteria (Psudomonas spp., Xanthomonas spp.), fungi (Magnaporthe oryzae, Tilletia barclyayana, etc.), oomycetes (Sclerophthora macrospora), phytoplasma (Candidatus phytoplasma 16srIII-L), virus (rice yellow mottle virus) and nematode (Aphelenchoides besseyi) on seeds.
Sorghum and millets: Some of the important sorghum seed-borne diseases are ergot (Claviceps sorghi), anthracnose (Colletotrichum graminicola), leaf blight (Exserohilum turcicum), downy mildew (Peronosclerospora sorghi), loose kernel smut (Sporisorium cruentum), long smut (S. ehrenbergii), head smut (S. reilianum), covered kernel smut (S. sorghi), bacterial blight (Ralstonia andropogoni), bacterial leaf streak (Xanthomonas vesicola pv. holcicola), and bacterial leaf spot (Pseudomonas syringae pv. syringae). Ergot (Claviceps fusiformis), and smut (Moesziomyces penicillariae) are the major seed-borne diseases of pearl millet. There are also some reports of downy mildew (Sclerospora graminicola) being seed-borne in nature. Melanopsichium eleusinis, Pyricularia grisea, and Bipolaris sp., are the important pathogens of small millet, for which salvaging treatment is used to recover pest-free seeds.
Wheat: The main risks associated with the germplasm exportation of bread and durum wheat are associated with pathogens, such as Karnal bunt (Tilletia indica), common bunt (T. tritici and T. laevis), Alternaria triticina, Xanthomonas translucens pv. undulosa, BSMV, and wheat streak mosaic virus (WSMV), which have a restricted geographical distribution. Nevertheless, many more wheat pathogens are listed in the requirements of the country importing the germplasm, and the measures taken to guarantee that the seeds are pathogen-free cover a wide range of possible threats by applying strict phytosanitary procedures in the multiplication field plots and exhaustive laboratory seed testing and seed treatments. Germplasm that is imported is subject to the NPPO regulations and inspected very carefully for wheat blast (Magnaporthe oryzae pathotype Triticum), dwarf bunt (T. controversa), and flag smut in wheat (Urocystis agropyri). In addition to the fungal pathogens, inspections are also carried for seed-borne insect pests, such as T. granarium (the khapra beetle), in seed exports and imports.
4.1.2. Grain and Oil Seed Legumes
Legume germplasm is more prone to pest attack, and many of these pests are known to spread through seeds [45
]. A list of regulatory pests and pathogens frequently tested in the legume germplasm regeneration sites of CGIAR is given in Supplementary Table S2
. The stringent phytosanitary and seed health testing procedures, such as those described for cereals, are also applied for legumes to prevent the transfer of fungal, bacterial, and viral diseases through legume germplasm. In general, germplasm and breeding lines for international transfers are regenerated under screenhouse conditions to avoid viral infections, and the germinated plants are inspected for viral symptoms and indexed by ELISA or PCR-based methods to ensure that plants are free from viruses prior to seed harvesting. Grow-out tests under screenhouse conditions are performed to assess seed-transmitted viruses, which is a standard practice for legumes. Although it is a time-consuming procedure, but it offers a reliable detection that eliminates the risk of viruses. Some of the important seed-borne and seed-transmitted pests, for which observations are conducted for export and import quarantine, are listed by crop below.
Bean: About 23 seed-borne bacterial, fungal, and viral pathogens are reported to be important for beans, including common blight (Xanthomonas campestris pv. phaseoli), charcoal rot (Macrophomina phaseolina), and anthracnose (Colletotrichum truncatum), along with three seed-transmitted viruses (alfaalfa mosaic virus (AMV), bean common mosaic virus (BCMV), and peanut mottle virus (PeMoV)).
Cowpea, bambara groundnut and other Vigna species: Several fungi and bacterial pathogens of cowpea are seed-borne, including cowpea bacterial blight (Xanthomonas axonopodis pv. vignicola), web blight (Rhizoctonia solani), and brown blotch (Colletotrichum capsici). About 10 viruses are reported to be seed-transmitted in cowpea. The most frequent viruses of interest in seed transmission are cucumber mosaic virus (CMV), cowpea yellow mosaic virus (CYMV), cowpea mottle virus (CmeV), southern bean mosaic virus (SBMV), and cowpea mild mottle virus (CPMMV). Cowpea seeds are subjected to fumigation with phostoxin (55% aluminum phosphide) to eliminate insect pests and treated with fungicide to eliminate seed-borne pathogens.
Chickpea and pigeonpea: Important seed-borne diseases of these two grain legumes are blight (Ascochyta rabiei), grey mold (Botrytis cinerea), wilt (Fusarium oxysporum f. sp. ciceri), and stem blight (Phomopsis longicolla) in chickpea; blight (Botryodiplodia theobromae) and wilt (Fusarium oxysporum f. sp. udum) in pigeonpea.
Faba bean: Twenty fungal species belonging to 13 genera were recognized as seed-borne risk (Aspergillus, Penicillium, Alternaria, Botrytis, Cephalosporium, Cladosporium, Epicoccum, Fusarium, Rhizoctonia, Rhizopus, Stemphylium, Trichothecium, and Verticillium), along with four seed-transmitted viruses [broad bean stain virus (BBSV), bean yellow mosaic virus (BYMV), broad bean mottle virus (BBMV), and pea seed-borne mosaic virus (PSbMV)]. Broomrape (Orobanche and Phelipanche spp.), root parasitic weeds, are also considered to pose a threat and measures are taken to avoid germplasm multiplication in the broomrape infested fields.
Groundnut: Dry root rot (M. phaseolina/Rhizoctonia bataticola), root rot (Rhizoctonia solani), pod rot (Sclerotium rolfsii), Sphaceloma arachidis (groundnut scab), Ralstonia solanacearum (African strains), seed bruchid (Stator pruininus), Testa nematode (Aphelenchoides arachidis), peanut mottle virus (PMV), peanut stripe virus (PStV), peanut clump virus (PCV), Indian peanut clump virus (IPCV), peanut stunt virus (PSV), and tobacco streak virus (TSV) are the important quarantine pests for groundnut.
Lentil: The important fungal seed-borne diseases of lentil include Ascochyta lentis (ascochyta blight), and Fusarium oxysporum f. sp. lentis (fusarium wilt), botrytis grey mold (Botrytis fabae and B. cinerea), Stemphylium blight (Stemphylium botryosum), phoma blight (Phoma medicaginis var. medicaginis), and anthracnose (C. lindemuthianu and C. truncatum); stem nematode (Ditylenchus dipsaci), and seed-transmitted viruses, include, AMV BYMV, PSbMV, CMV, and BBSV.
Soybean: The seed-borne fungal and bacterial pathogens of soybean are soybean bacterial pustule (X. axonopodis pv. glycinea), brown spots (Septoria glycinea), frogeye leaf spots (Cercospora sojina), yellow leaf spots (P. manshurica), charcoal rot (M. phaseolina), and anthracnose (C. truncatum). Many seed-transmitted viruses are also reported in soybean, including BCMV, CMV, CYMV, CmeV, CPMMV, and SBMVin West Africa. Rigorous tests are also conducted for other viruses depending on the country of origin. Seeds are fumigated with phostoxin (55% aluminum phosphide) to eliminate insect pests and fungicide treatments are given to eliminate seed-borne pathogens.
4.2. Vegetatively Propagated Crops
Banana (and plantain), cassava, potato, sweetpotato, and yam are the major vegetatively propagated crops (VPCs) exchanged by the CGIAR programs [55
]. Vegetatively propagation poses the greatest risk of the introduction of pests through planting material, which can carry any infections from previous seasons to the next cropping cycle and thus accumulate pathogens, especially viruses, over generations of cultivation. Many transboundary pest introductions have been linked with the transfer of vegetative propagules: the spread of BBTV to Africa and its further spread in the continent [19
]; in the case of potato, the necrotic strains of potato virus Y (PVY) in Brazil and aggressive strains of potato late blight in Africa and Asia and potato cyst nematode (Globodera palladi
) in East Africa; in the case of cassava, the regional spread of cassava brown streak virus (CBSV), which is attributed to contaminated stem propagation; and in Asia, the spread of Sri Lankan cassava mosaic virus (SLCMV) from South Asia to East Asia [57
]. Therefore, many countries regulate vegetative germplasm importation, and the FAO-IPGRI technical guidelines recommend that only in vitro plants that have been tested for pathogens should be moved between countries [36
]. The pollen or true seed of these crops are also exchanged for breeding purposes under adequate phytosanitary controls. By limiting international movement to sterile in vitro plants, the only concern that remains is intracellular obligate pathogens, such as viruses, viroids, and phytoplasmas. Depending on the country, some viruses are regulated by quarantine procedures (e.g., BBTV, CBSV, and PVY), and several other viruses are unregulated (e.g., sweet potato mild mosaic virus). Nonetheless, the standard procedure used by GHUs includes the generation of virus-free in vitro plants as per the FAO/IBPGR technical guidelines for the conservation and distribution of these crops [36
]. All the material exported and imported are tested for viruses, and other pests under NPPO guidance, and only material free of viruses, and other pests, is released to the end-users. Unlike cereals and legumes, the phytosanitation, and testing procedures for clonally propagated crops differ according to the crop species, as explained below.
Banana is a perennial herbaceous plant, traditionally propagated using suckers (side shoots generated from underground corms), and is thus often carries both soil-borne insects and fungi, in addition to shoot-invading viruses, fungi, and bacterial agents. Several banana pathogens, like Fusarium oxysporum
f. sp. cubense
tropical race 4 (Panama disease), banana Xanthomonas wilt (Xanthomonas campestris pv. musacearum
), and several viruses, such as BBTV and banana bract mosaic virus (BBrMV) have restricted geographic distribution. Guaranteeing the movement of pathogen-free germplasm is an important task to minimize the risk of these regulated quarantine pest introduction into new countries. Pathogens that are often symptomless in germplasm (e.g., in vitro plants, corms, and suckers), such as viruses, pose a special risk to the movement of vegetative germplasm. The Bioversity International-CIAT Alliance (1617 accessions), and the IITA (393 accessions) germplasm collections are managed as in vitro cultures. It has been shown that bacterial and fungal contaminants in banana shoot tip culture can be eradicated by isolating small explants, e.g., 1 mm meristems, and culturing them in vitro, but the virus infection still presents an important risk. To mitigate these risks, the Conservation Thematic Group of MusaNet, an international network for Musa
genetic resources coordinated by Bioversity International, has recently edited a new version of technical guidelines to minimize the risk of pest introductions, through the movement of germplasm [58
]. These guidelines followed a recommendation issued on the basis of an analysis of the phytosanitary procedures carried out by GHUs [56
]. As per the new guidelines, at least four plants for each accession are grown for six months in a greenhouse. Leaf sampling is carried out from the limb and midrib of the three youngest leaves after 3 and 6 months for the comprehensive detection of the five most important viruses by PCR/RT-PCR: BBrMV, BBTV, banana streak virus (BSV), banana mild mosaic virus (BanMMV), and cucumber mosaic virus (CMV). Comprehensive indexing using electron microscopy is also conducted to search for any viral particle. Sanitation of the virus-infected banana accession is a complex process requiring a combination of meristem culturing, thermotherapy, and chemotherapy. Despite numerous efforts and the continuous optimization of the protocols, the success rate of banana sanitation is around 70%. An accession indexed negative is added to an in vitro banana collection for further safe propagation and distribution. All precautions are taken to avoid any further infection to in vitro plants that could arise if the plant is transferred to the field or greenhouse before distribution.
One of the major challenges for banana germplasm exchange was posed by the finding of an integration of the BSV genome, termed the eBSV (endogenous BSV), in the M. balbisiana
genome, which contributes to the B genome. The eBSV can spontaneously release infectious particles, especially following in vitro culturing and interspecific crosses [19
]. The presence of infectious eBSVs within B genomes has emerged as a main constraint for health indexing and safe Musa
germplasm transfers. Plants apparently negative to BSV could spontaneously become positive with the expression of the eBSV sequence. The discovery of this phenomenon in bananas in the 1990s halted banana germplasm distribution from CGIAR centers. However, the Inter-African Phytosanitary Council, the Regional Plant Protection Organization of Africa, made a provision allowing the distribution within Africa of virus-free banana and plantain that may carry eBSV [19
]. Based on this regulation, the IITA genebank and breeding programs distribute virus-free banana germplasm within Africa, with the informed consent of the recipients. However, the advancement of technology and knowledge on viruses integrated in host genomes provide a way to overcome this natural bottleneck to germplasm distribution. First, diagnostic techniques were established to distinguish eBSV and episomal virus particles for virus indexing purposes; secondly, molecular markers were established to identify Musa
accessions with activatable eBSV; and lastly, a decision model was developed to enable the distribution of Musa
germplasm with eBSV sequences based on the consent of the importer [58
Cassava is cultivated for tuberous roots and is traditionally propagated using stem cuttings. The crop is conserved in field collections and in vitro. The in vitro collection of 6500 accessions of cassava at the CIAT in Colombia, and 3700 accessions at the IITA in Nigeria are the largest cassava ex situ collections. The germplasm is exchanged as in vitro plants and botanic seed. Viruses and phytoplasmas pose a major threat to cassava distribution as in vitro plants. A diverse range of viruses infect cassava in Latin America, Africa, and Asia (Supplementary Table S1
]. The sanitary testing of the cassava collection held by the CIAT in Colombia checks for viruses prevalent in the region: Cassava common mosaic virus (CsCMV), cassava virus X (CsXV), and four other viruses that are associated with the cassava frogskin disease: the cassava frogskin associated virus (CsFSaV), cassava polero-like virus (CsPLV), cassava new alphaflexivirus (CsNAV), and cassava torrado-like virus (CsTLV) [60
]. The sanitary testing of the in vitro African cassava collection conserved in the IITA, Nigeria, mainly checks for viruses prevalent in Africa: African cassava mosaic virus (ACMV), a complex of East African cassava mosaic viruses (EACMVs), and its strains, cassava brown streak ipomoviruses (CBSIVs), and 16Sr Phytoplasmas [57
]. The cassava collection in Asia mainly focuses on viruses (Indian cassava mosaic virus (ICMV), and SLCMV), and phytoplasmas prevalent in the region. Both the CIAT and IITA GHUs have the diagnostic capability to test for all viruses known to infect cassava.
Several procedures have been established for virus and phytoplasma detection to generate virus-free planting material from meristem cultures in vitro, with or without thermotherapy, chemotherapy, or cryotherapy. The basic procedure includes a heat treatment applied to of stem cuttings with a length of about 30 at 28 and 38 °C for 6 h in the dark and 18 h in the light in an incubator [43
]. Apical shoots from stem cuttings, after sanitation with 3% sodium hypochlorite, are used for meristem excision and in vitro plant development. About 2- to 4-month-old in vitro plants are virus indexed by PCR or RT-PCR to detect and eliminate virus-infected plants, and the remaining plants are re-indexed second time after 3 to 4 months to ascertain their health status. The virus-free plants are used as a mother stock for conservation as ‘clean stock’, and further propagation and use. It takes about 6 to 12 months to generate a virus-free stock of cassava germplasm. These procedures are known to be robust and 90% efficient in eliminating viruses. Only virus-free in vitro plants are transferred for propagation purposes. Cassava germplasm distributed as botanic seed poses little risk of virus spread, as none of the viruses reported to infect cassava have been detected in seedlings. Nonetheless, the cassava botanic seeds are surface sterilized with insecticides and pesticides, and they are germinated in screenhouses for physical inspection, before the seedlings are released to the end-users. Occasionally, cassava germplasm is transferred as stem cuttings generated from virus-free in vitro plants under insect-proof screenhouse conditions, after stem treatment with a slurry of insecticide and fungicide cocktail to eliminate microorganisms and arthropod pests.
Potato is propagated through tubers. Besides viruses, viroids, and phytoplasmas, field-produced tubers can transmit a long list of bacterial, fungal, and nematode diseases, including brown rot (Ralstonia solanacearum phylotype IIB
), softrot (Pectobacterium
spp.) ringrot (Clavibacter michiganensis
), wart (Synchytrium endobioticum
), common scab (Streptomyces
sp.), powdery scab (Spongospora subterranea
f. sp. subterranea
), late blight (Phytophthora infestans
), nematodes, and insects. Potato is known to be infected by more than 50 different viruses, but only about a handful of them (PVY, PVX, PVS, PVA, and potato leaf roll virus) are significant pathogens globally; however, some unique local viruses can be of major concern [61
]. The CIP maintains one of the world’s largest in-vitro genebank collections with over 7209 accessions of potato, many of which are of a local origin. The CIP uses a ISO/IEC17025 accredited process for ensuring that in vitro plants are free of all pathogens, both known and unknown. The process includes a combination of an antibacterial treatment before and during in vitro introduction, followed by virus indexing, which combines ELISA (9 viruses), RT-PCR (1 virus and 1 viroid), and a biological indicator host infection for 11 species. This indexing is repeated twice, before and after thermo-therapy and meristem tip culturing. Due to the extremely contagious and stable nature of PSTVd all plants are tested for this viroid before even starting the process of introduction and are destroyed immediately if they are found to be positive. The protocol for virus cleaning at the CIP is 91% efficient, and the most difficult viruses to clean are PVS and PVT. Only material that has been certified to be free of any pathogens after this process is permitted to be moved internationally. Any germplasm received from other institutions or countries will be tested in a similar way, before it can be made available for further distribution. Despite the rigorous indexing process, additional diagnostic tests are performed for pathogens when demanded by the importing country. Breeders occasionally move true seed and pollen internationally, and this is generally considered to be safer than moving plants or organs around. For true seed and pollen, the procedure includes testing both parents (female and male) at the pre-flowering stage for seed-transmitted viruses (APLV, APMMV, AVB-O, AMV, PVT, and PYV) and PSTVd, for which they must be negative. The seeds and pollen must be free of any insect pests and treated at −70 °C for seven days, if contamination is suspected. The seeds are surface sterilized with 2.5% sodium hypochlorite for 10 min to kill any seed-borne pathogens.
Sweetpotato is traditionally multiplied through stem (vine) cuttings. Like other VPCs, viruses are the principal concern for vegetatively propagation. More than 30 viruses have been reported to infect sweetpotato, but only a handful of them are of global significance and include crinivirus, sweet potato chlorotic stunt virus (SPCSV), the potyviruses, sweet potato feathery mottle virus (SPFMV), sweet potato virus C (SPVC), -G (SPVG), and -2 (SPV2), and several related begomoviruses (Supplementary Table S2
]. The CIP genebank holds 8,054 accessions of sweetpotato, and like potato, CIP has a ISO/IEC17025 accredited process for ensuring that in vitro cultivated sweetpotato are free of all known pathogens. The process is similar to that described for potato, except that testing is conducted for sweetpotato-specific viruses by PCR (begomoviruses) and ELISA (10 viruses), and that the biological indicator host range is replaced by a single universal indicator host, Ipomoea setosa
, on which sweetpotato accessions are grafted. The efficiency of the current virus clean-up protocols for sweetpotato is 69%, and the difficult viruses to eliminate are begomoviruses, SPFMV, and SPVG. As for potatoes, only in vitro plants that have been confirmed to be free of known pathogens are distributed internationally. Sweetpotato pollen or seed is not commonly moved internationally by the CIP or partners, but the procedure would be similar to that for potato. Only two sweetpotato viruses, a begomovirus (sweet potato leaf curl virus), and a mastrevirus (sweet potato symptomless virus 1), have been reported to be seed-transmitted.
: Unlike other clonal crops, multiple species of yam (Dioscorea
spp.), which originated from different parts of the world, have been domesticated for the production of underground edible tubers, and the crop is traditionally propagated using tubers [64
]. Out of about ten popularly grown species, D. rotundata
(white yam) of West African origin, and D. alata
(water yam) of Asiatic origin are widely cultivated in the world. The IITA genebank in Nigeria holds the world’s largest in vitro yam collection [64
]. The global yam germplasm collection comprises about 5839 accessions of about 10 species, including D. rotundata
, D. alata, D. bulbifera, D. cayennensis, D. dumetorum, D. esculenta,
and a few other species. Yam is known to be infected by over 15 viruses [57
], including yam mosaic virus (YMV), CMV, yam mild mosaic virus (YMV), several badnaviruses, generically referred to as yam bacilliform viruses (YBVs), Japanese yam mosaic virus, Chinese yam necrotic mosaic virus, yam asymptomatic virus 1, yam virus Y, yam chlorotic necrosis virus (YCNV), yam chlorotic mosaic virus (YCMV), Dioscorea mosaic-associated virus, and air potato ampelovirus 1. Of these, YMV is known to cause the most economic damage in D. rotundata
and D. cayanensis
worldwide, whereas YCMV and YCN are important for Chinese and Japanese yam in Asia. The other viruses detected in yam either cause mild mottling or no symptoms at all. Many yam viruses are not regulated, although the IITA uses protocols to generate virus-free in vitro plants for conservation and distribution [66
]. Virus elimination is achieved by selecting asymptomatic plants for thermotherapy and regeneration of in vitro plants from meristem explants. The in vitro plants are subjected to virus indexing using PCR-based methods, after three months of post-flask growth, to ascertain their health status. Plants that test negative are bulk propagated for conservation and distribution. The integrated viral sequences of badnaviruses have been detected in yam genome sequences, but unlike in eBSV in bananas, the endogenous badnavirus sequences in yam are defective and not known to generate infectious particles. They were therefore not regarded to have any phytosanitary significance. Compared to other VPCs conserved by the CGIAR, yam phytosanitation is a lengthy process and usually takes between 12 to 24 months. Of the various associated factors, the slow in vitro meristem growth is a major bottleneck for the production of virus-free plants. True seed of yam is exchanged by breeding programs after seed treatment with a fungicide and an insecticide. Tubers generated from virus-free yam plants are also used for international exchange, especially within the West African sub-region, by breeding and seed system initiatives.