Phytosanitary Interventions for Safe Global Germplasm Exchange and the Prevention of Transboundary Pest Spread: The Role of CGIAR Germplasm Health Units.

The inherent ability of seeds (orthodox, intermediate, and recalcitrant seeds and vegetative propagules) to serve as carriers of pests and pathogens (hereafter referred to as pests) and the risk of transboundary spread along with the seed movement present a high-risk factor for international germplasm distribution activities. Quarantine and phytosanitary procedures have been established by many countries around the world to minimize seed-borne pest spread by screening export and import consignments of germplasm. The effectiveness of these time-consuming and cost-intensive procedures depends on the knowledge of pest distribution, availability of diagnostic tools for seed health testing, qualified operators, procedures for inspection, and seed phytosanitation. This review describes a unique multidisciplinary approach used by the CGIAR Germplasm Health Units (GHUs) in ensuring phytosanitary protection for the safe conservation and global movement of germplasm from the 11 CGIAR genebanks and breeding programs that acquire and distribute germplasm to and from all parts of the world for agricultural research and food security. We also present the challenges, lessons learned, and recommendations stemming from the experience of GHUs, which collaborate with the national quarantine systems to export and distribute about 100,000 germplasm samples annually to partners located in about 90 to 100 countries. Furthermore, we describe how GHUs adjust their procedures to stay in alignment with evolving phytosanitary regulations and pest risk scenarios. In conclusion, we state the benefits of globally coordinated phytosanitary networks for the prevention of the intercontinental spread of pests that are transmissible through plant propagation materials.

Efficiency of insect-proof net tunnels in reducing virus-related seed degeneration in sweet potato.

Virus-related degeneration constrains production of quality sweet potato seed, especially under open field conditions. Once in the open, virus-indexed seed is prone to virus infection leading to decline in performance. Insect-proof net tunnels have been proven to reduce virus infection under researcher management. However, their effectiveness under farmer-multiplier management is not known. This study investigated the ability of net tunnels to reduce degeneration in sweet potato under farmer-multiplier management. Infection and degeneration were assessed for two cultivars, Kabode and Polista, grown in net tunnels and open fields at two sites with varying virus pressures. There was zero virus incidence at both sites during the first five generations. Sweet potato feathery mottle virus and sweet potato chlorotic stunt virus were present in the last three generations, occurring singly or in combination to form sweet potato virus disease. Virus infection increased successively, with higher incidences recorded at the high virus pressure site. Seed degeneration modelling illustrated that for both varieties, degeneration was reduced by the maintenance of vines under net tunnel conditions. The time series of likely degeneration based on a generic model of yield loss suggested that, under the conditions experienced during the experimental period, infection and losses within the net tunnels would be limited. By comparison, in the open field most of the yield could be lost after a small number of generations without the input of seed with lower disease incidence. Adopting the technology at the farmer-multiplier level can increase availability of clean seed, particularly in high virus pressure areas.

The complete genome sequences of two isolates of potato black ringspot virus and their relationship to other isolates and nepoviruses.

The complete nucleotide sequences of RNA 1 and RNA 2 of the nepovirus potato black ringspot virus (PBRSV) from two different isolates were determined, as well as partial sequences from two additional isolates. RNA1 is 7,579-7,598 nucleotides long and contains one single open reading frame (ORF), which is translated into a large polyprotein with 2,325 amino acids and a molecular weight of 257 kDa. The complete sequence of RNA2 ranges from 3857 to 3918 nt between the different isolates. It encodes a polyprotein of 1079-1082 amino acids with a molecular weight of 120 kDa. Sequence comparison using the Pro-Pol region and CP showed that all four isolates formed two distinct groups, corresponding to potato and arracacha, that were closely related to each other and also to tobacco ringspot virus (TRSV). Comparing our data to those obtained with other nepoviruses, our results confirm that PBRSV belongs to a distinct species and is a member of subgroup A in the genus Nepovirus based on its RNA2 size, genome organization, and nucleotide sequence.

Some molecular characteristics of three viruses from SPVD-affected sweet potato plants in Egypt.

Sweet potato feathery mottle virus (SPFMV, genus Potyvirus, family Potyviridae), Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus, family Closteroviridae) and sweet potato virus G (SPVG) were detected in naturally infected sweet potato plants grown in the Delta region in Egypt. Before this study, SPVG was reported only from China. Two isolates of SPFMV and one isolate of SPVG were characterized for the 3'-proximal genomic sequences. Phylogenetic analyses indicated that the SPFMV isolates belong to the "russet crack" strain group (RC). Serological tests using monoclonal antibodies, and phylogenetic analysis of a partial sequence of the Hsp70 gene, indicated that the Egyptian SPCSV belongs to the so-called non-East African strain group of SPCSV.

Coat protein sequence analysis reveals occurrence of new strains of Sweet potato feathery mottle virus in Uganda and Tanzania.

The 3'-proximal part (1.8 kb) of the Sweet potato feathery mottle virus (SPFMV) genome was studied in four SPFMV isolates collected from farmers' fields in western Uganda (SPFMV-Bny), eastern Uganda (SPFMV-Sor) and Bagamoyo district, Tanzania (SPFMV-TZ1 and SPFMV-TZ2). Unlike the other three SPFMV isolates, SPFMV-Sor was not detected with the polyclonal antisera to SPFMV. It showed moderately high coat protein (CP) nucleotide (93.3-96.7%) and amino acid (93.6-96.8%) sequence identity to the isolates of the SPFMV strain group C. In contrast, identities (78.1-80.1%, and 79.9-83.1%) to isolates of the SPFMV strain groups O, RC, and the East African (EA) strain group were low. Similar to some isolates (SPFMV-CH2 and SPFMV-6) of strain group C, but different from other SPFMV isolates, SPFMV-Sor contained a deletion of 6 nucleotides in the CP-encoding region (CP amino acid positions 62-63). Phylogenetic analysis of the CP sequences indicated that SPFMV-Sor belongs to the SPFMV strain group C that has not been reported from Africa. Sequence data were obtained for the first time from Tanzanian SPFMV isolates in this study, and phylogenetic analysis indicated that they belong to the strain group EA, which is unique to East Africa.

Complete genome sequence and analyses of the subgenomic RNAs of sweet potato chlorotic stunt virus reveal several new features for the genus Crinivirus.

The complete nucleotide sequences of genomic RNA1 (9,407 nucleotides [nt]) and RNA2 (8,223 nt) of Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) were determined, revealing that SPCSV possesses the second largest identified positive-strand single-stranded RNA genome among plant viruses after Citrus tristeza virus. RNA1 contains two overlapping open reading frames (ORFs) that encode the replication module, consisting of the putative papain-like cysteine proteinase, methyltransferase, helicase, and polymerase domains. RNA2 contains the Closteroviridae hallmark gene array represented by a heat shock protein homologue (Hsp70h), a protein of 50 to 60 kDa depending on the virus, the major coat protein, and a divergent copy of the coat protein. This grouping resembles the genome organization of Lettuce infectious yellows virus (LIYV), the only other crinivirus for which the whole genomic sequence is available. However, in striking contrast to LIYV, the two genomic RNAs of SPCSV contained nearly identical 208-nt-long 3' terminal sequences, and the ORF for a putative small hydrophobic protein present in LIYV RNA2 was found at a novel position in SPCSV RNA1. Furthermore, unlike any other plant or animal virus, SPCSV carried an ORF for a putative RNase III-like protein (ORF2 on RNA1). Several subgenomic RNAs (sgRNAs) were detected in SPCSV-infected plants, indicating that the sgRNAs formed from RNA1 accumulated earlier in infection than those of RNA2. The 5' ends of seven sgRNAs were cloned and sequenced by an approach that provided compelling evidence that the sgRNAs are capped in infected plants, a novel finding for members of the Closteroviridae.

Comparisons of coat protein gene sequences show that East African isolates of Sweet potato feathery mottle virus form a genetically distinct group.

Sweet potato feathery mottle virus (SPFMV, genus Potyvirus) infects sweet potatoes (Ipomoea batatas) worldwide, but no sequence data on isolates from Africa are available. Coat protein (CP) gene sequences from eight East African isolates from Madagascar and different districts of Uganda (the second biggest sweet potato producer in the world) and two West African isolates from Nigeria and Niger were determined. They were compared by phylogenetic analysis with the previously reported sequences of ten SPFMV isolates from other continents. The East African SPFMV isolates formed a distinct cluster, whereas the other isolates were not clustered according to geographic origin. These data indicate that East African isolates of SPFMV form a genetically unique group.

Transgenic resistance to PVY(O) associated with post-transcriptional silencing of P1 transgene is overcome by PVY(N) strains that carry highly homologous P1 sequences and recover transgene expression at infection.

Resistance to Potato virus Y (PVY) has been obtained in our previous studies through expression of the PVY P1 gene in sense or antisense orientation in potato cv. Pito. In the present study, the mechanism and strain specificity of the resistance were analyzed. Several features including low steady-state P1 mRNA expression in the resistant P1 plants indicated that resistance was based on post-transcriptional gene silencing (PTGS). Resistance was specific to PVY(O) isolates, the PVY strain group from which the P1 transgene was derived. However, according to group analyses, there was no distinguishing characteristic between the PVY(O) and PVY(N) strains P1 gene sequences. Therefore, the ability of the PVY(N) strains to overcome resistance could not be explained solely based on their P1 gene sequences. Infection with PVY(N) of the PVY(O)-resistant transgenic lines led to a recovery of expression of the P1 transgene. These data suggested that factors other than sequence homology are required in determination of the resistance specificity.

Synergistic interactions of a potyvirus and a phloem-limited crinivirus in sweet potato plants.

When infecting alone, Sweet potato feathery mottle virus (SPFMV, genus Potyvirus) and Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus) cause no or only mild symptoms (slight stunting and purpling), respectively, in the sweet potato (Ipomoea batatas L. ). In the SPFMV-resistant cv. Tanzania, SPFMV is also present at extremely low titers, though plants are systemically infected. However, infection with both viruses results in the development of sweet potato virus disease (SPVD) characterized by severe symptoms in leaves and stunting of the plants. Data from this study showed that SPCSV remains confined to phloem and at a similar or slightly lower titer in the SPVD-affected plants, whereas the amounts of SPFMV RNA and CP antigen increase 600-fold. SPFMV was not confined to phloem, and the movement from the inoculated leaf to the upper leaves occurred at a similar rate, regardless of whether or not the plants were infected with SPCSV. Hence, resistance to SPFMV in cv. Tanzania was not based on restricted virus movement, neither did SPCSV significantly enhance the phloem loading or unloading of SPFMV. It is also noteworthy that SPVD is an unusual synergistic interaction in that the potyvirus component is not the cause of synergism but is the beneficiary. It is hypothesized that SPCSV is able to enhance the multiplication of SPFMV in tissues other than where it occurs itself, perhaps by interfering with systemic phloem-dependent signaling required in a resistance mechanism directed against SPFMV.

Two Serotypes of Sweetpotato feathery mottle virus in Uganda and Their Interaction with Resistant Sweetpotato Cultivars.

ABSTRACT Isolates of Sweetpotato feathery mottle virus (SPFMV, genus Potyvirus, family Potyviridae) were obtained in several districts of Uganda from sweetpotato plants infected with the sweetpotato virus disease (SPVD), the most important disease of this crop in Africa. A monoclonal antibody (MAb 7H8) raised against the coat proteins (CP) of a mixture of the SPFMV strain C (United States) and the isolate SPV-I (West Africa) distinguished Ugandan SPFMV isolates into those detectable and not detectable by the MAb. These two serotypes differed in prevalence in different districts of Uganda and in two common sweetpotato cultivars. Both serotypes could be transmitted simultaneously by single aphids. The serotypes differed in their ability to systemically coinfect sweetpotatoes that were infected with Sweetpotato chlorotic stunt virus (SPCSV, genus Crinivirus), the virus required to induce SPVD in SPFMV-infected plants. One sweetpotato breeding line, resistant to SPFMV from the New World, was infected by graft-inoculation with all SPFMV isolates from Uganda. Another SPFMV-resistant sweetpotato line became infected with SPFMV and developed SPVD only following coinoculation with SPCSV.

Phylogenetic Analysis of 16S rRNA Genes and PCR Analysis of the nec1 Gene from Streptomyces spp. Causing Common Scab, Pitted Scab, and Netted Scab in Finland.

ABSTRACT The sequences of the 16S rRNA genes (nucleotides 29 to 1,521) from various Streptomyces strains pathogenic to potato were compared. These included 10 pathogenic Streptomyces strains isolated from potato scab lesions in Finland, the type strains of S. aureofaciens NRRL 2209(T) and S. lydicus ATCC 25470(T), 'S. griseus subsp. scabies' ATCC 10246, and two S. griseus strains that were originally deposited to the collection as pathogens. The nucleotide sequence (>94.5% sequence identity [SI]) and length (1,469 to 1,481 nucleotides) of the analyzed region varied. Phylogenetic analysis of 16S rRNA genes placed Finnish strains into three species, supported by previously characterized morphological and physiological traits. Six Finnish strains, including two strains that deviated from the others in one trait (no spiral sporophores or D-xylose utilization), had identical 16S rRNA genes and were identified as S. scabies (99.9% SI to S. scabies ATCC 49173). Three Finnish strains were identified as S. turgidiscabies, a species previously described only in Japan (99.9% SI to S. turgidiscabies ATCC 700248). Finnish strain 317 and S. aureofaciens NRRL 2209 (99.8% SI) were placed in a distinct phylogenetic cluster together with Kitosatospora spp., which suggests that S. aureofaciens may belong to the recently revived genus Kitosatospora. In pathogenicity tests, S. scabies caused characteristic symptoms of common scab, S. turgidiscabies caused mainly pitted scab, and S. aureofaciens caused netted scab and necrotic lesions on stolons of potato cultivars Bintje and Matilda in the greenhouse. The nec1 gene and the intergenic region between nec1 and the 5' transposase pseudogene ORFtnp were successfully amplified by polymerase chain reaction from S. scabies ATCC 49173 and the pathogenic Finnish strains of S. scabies, but not from a nonpathogenic strain of S. scabies, three pathogenic and two nonpathogenic strains of S. turgidiscabies, and S. aureofaciens.

Biological, serological, and molecular differences among isolates of potato a potyvirus.

ABSTRACT Sequences of the coat protein (CP) and 3'-end nontranslated region (3'NTR) of 13 isolates and the helper component proteinase (HC) of nine isolates of potato A potyvirus (PVA) were determined and compared with the eight previously determined PVA CP and 3'NTR sequences and one HC sequence. CP amino acid (aa), 3'NTR nucleotide, and HC aa sequence identities were 92.9, 93.4, and 94.8%, respectively. Sequence data, serological tests, and the necrotic local lesions induced in the leaves of the potato hybrid 'A6' confirmed that tamarillo mosaic virus is a strain of PVA. The aa substitutions A6T and G7S in the CP N-terminus were correlated with loss of aphid transmissibility. Development of necrotic lesions or nonnecrotic symptoms in the systemically infected leaves or lack of systemic spread in potato cv. King Edward were used to place the PVA isolates into four strain groups, but this grouping was not correlated with any differences in CP, HC, or 3'NTR. Recognition of CP by three monoclonal antibodies was used to place the PVA isolates into three groups different from the four groups above. The epitopes of two mono-clonal antibodies were mapped by site-directed mutagenesis to the same lysine residue at the CP aa 34.