Infected Propagation Material Research Articles

First report of citrus yellow vein clearing virus (CYVCV) on citrus in Italy.

In March 2024, symptoms of vein clearing (Fig. S1) were observed on leaves of six lemon (Citrus limon (L.) Burm. f.) trees grown in a private garden in Palma Campania (Campania region, Italy). Symptoms strongly resembled those of yellow vein clearing disease, caused by Potexvirus citriflavivenae (citrus yellow vein clearing virus, CYVCV), genus Potexvirus, sub-genus Mandarivirus (Loconsole et al., 2012; Cui et al., 2018). CYVCV was reported in Pakistan, India, China, Iran, Turkey, Korea and USA (Abrahamian et al., 2024; Catara et al., 1993; EPPO, 2024; Sun and Yokomi, 2024; Jin et al., 2024). Since 2022, considering the potential impact of this emerging virus, CYVCV has been included in the EPPO alert list and it has not been reported in any European Union (EU) Member State where the virus is not regulated. To ascertain possible infection with CYVCV, leaf samples were collected and tested by reverse transcription polymerase chain reaction (RT-PCR). Total nucleic acids (TNAs) from leaves of the symptomatic lemon and non-symptomatic lemon and sweet orange (Citrus sinensis (L.) Osbeck) trees grown in the same garden, were extracted using the silica-capture extraction method (Foissac et al. 2001). TNAs were tested by RT-PCR using CYVCV specific primers (Table S1) designed by Chen et al., (2014) targeting the coat protein gene. All symptomatic trees and an asymptomatic sweet orange from the same garden tested positive generating an amplicon of the expected size (612 bp). Direct Sanger sequencing of amplicons obtained from two symptomatic lemons and one non-symptomatic sweet orange (An PP842725-PP842727) followed by BLAST search showed 97.9-98.08% sequence identity (query coverage 100%) with CYVCV isolates previously reported from a mandarin (An OQ418501) and a lemon (An OQ418493) in California. In contrast, no amplicons were detected in samples from non-symptomatic lemon trees. Virus identity was confirmed by RT-PCR with different primers (Table S1) targeting the RNA-dependent RNA polymerase gene of CYVCV. Amplicons of the expected size (832 bp) were obtained only from samples previously tested positive to the virus and their sequences determined by Sanger sequence (An PQ284953-PQ284955) showed 97.98-98.11% identity (query coverage 100%) with CYVCV sequence previously reported from a mandarin (An OR251443) in New Delhi. Since CYVCV is transmitted by insects (Önelge et al., 2011a, 2011b, Zhang et al., 2018, 2019), TNAs (Foissac et al., 2001) from Aphis aurantii infesting symptomatic lemons, and of A. spiraecola and Dialeurodes citri infesting symptomatic lemon and non-symptomatic sweet orange trees were assayed by RT-PCR with primer pairs designed by Chen et al., (2014). Three samples of 15 specimens from three different trees were tested for each insect. In contrast to D. citri, which tested negative, an amplicon of 612 bp was detected from A. aurantii and A. spiraecola and Direct sequencing of amplicons (An PP842721-PP842724) confirmed the nature of the virus and showed that insect-derived viral sequences shared nucleotide identity of 99.3-100% with those from the infected trees. Altogether, these data support that CYVCV is present in Southern Italy. Likely, it has been introduced illegally through infected citrus propagation material. Given the large A. aurantii and A. spiraecola populations in the areas where CYVCV has been detected, a fast spread of the virus to other areas of Campania region can be expected. Even if the impact of CYVCV on citrus production in Italy remains to be assessed, prompt and extensive surveys may be helpful to establish the actual distribution of the virus. This would allow for a better evaluation of appropriate measures to be adopted to achieve sustainable citrus production. At the best of our knowledge, this is the first report of CYVCV in Italy and in the EU territory.

A streak through history: Solving the riddle of chlorotic streak disease of sugarcane

AbstractUntil recently, the identity of the pathogen of chlorotic streak disease (CSD) was one of the most enduring mysteries of sugarcane pathology. The mystery continued when the causal agent was revealed as a member of a large group of free‐living eukaryotic microbes that had not previously been associated with any plant diseases. CSD has impacted worldwide sugarcane production since at least the 1920s when it was first noticed simultaneously in Java, Australia and Hawaii. Readily identified by irregular, yellow to creamy‐white chlorotic streaks on the leaves, the identity of the pathogen remained unknown for 90 years. CSD negatively affects germination speed, ratooning, stalk numbers and stalk weight, and later causes stunting of crop growth, leading to major losses in sugar production. Prior to the discovery of the pathogen, CSD was found to be waterborne and spread by infected vegetative propagation material. As such it is particularly damaging in poorly drained soils and areas subject to high rainfall. In severe cases it can lead to crop death. The implementation of successful CSD management strategies has been limited by the lack of knowledge surrounding the nature of this disease. However, these strategies are likely to be augmented with the recent identification of the causal agent, a novel cercozoan described as Phytocercomonas venanatans. This review provides insight into the history, causal agent and potential future developments of CSD management strategies.

Evolutionary Analysis of Grapevine Virus A: Insights into the Dispersion in Sicily (Italy)

Grapevine virus A (GVA) is a phloem-restricted virus (genus Vitivirus, family Betaflexiviridae) that cause crop losses of 5–22% in grapevine cultivars, transmitted by different species of pseudococcid mealybugs, the mealybug Heliococcus bohemicus, and by the scale insect Neopulvinaria innumerabilis. In this work, we studied the genetic structure and molecular variability of GVA, ascertaining its presence and spread in different commercial vineyards of four Sicilian provinces (Italy). In total, 11 autochthonous grapevine cultivars in 20 commercial Sicilian vineyards were investigated, for a total of 617 grapevine samples. Preliminary screening by serological (DAS-ELISA) analysis for GVA detection were conducted and subsequently confirmed by molecular (RT-PCR) analysis. Results showed that 10 out of the 11 cultivars analyzed were positive to GVA, for a total of 49 out of 617 samples (8%). A higher incidence of infection was detected on ‘Nerello Mascalese’, ‘Carricante’, ‘Perricone’ and ‘Nero d’Avola’ cultivars, followed by ‘Alicante’, ‘Grecanico’, ‘Catarratto’, ‘Grillo’, ‘Nerello Cappuccio’ and ‘Zibibbo’, while in the ‘Moscato’ cultivar no infection was found. Phylogenetic analyses carried out on the coat protein (CP) gene of 16 GVA sequences selected in this study showed a low variability degree among the Sicilian isolates, closely related with other Italian isolates retrieved in GenBank, suggesting a common origin, probably due to the exchange of infected propagation material within the Italian territory.

Monitoring the distribution of banana bunchy top virus in South Africa: a country-wide survey.

Banana bunchy top disease is the most devastating viral disease of bananas worldwide and is caused by banana bunchy top virus (BBTV). The disease is spread by the banana aphid Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae) and through infected propagation material. In 2016, the virus was detected for the first time in an isolated area in the South Coast region of KwaZulu-Natal Province (KZN), South Africa. The aim of this study was to conduct surveys across all banana-producing regions in South Africa, viz. KwaZulu-Natal, Mpumalanga, and Limpopo provinces. Over 1700 plant and aphid samples were collected from commercial farms and rural households in the three provinces, and more-intense sampling was done in the affected KZN region. A BBTV-specific PCR targeting DNA-R (encoding the master replication initiation protein, M-Rep) was used to detect virus-infected samples, and amplicons of the expected size were sequenced. Comparative phylogenetic analysis showed that the South African BBTV isolates clustered within the Pacific Indian Oceans genomic group, which includes isolates from India and other regions in Africa, with a bootstrap value of 94%. To date, the virus has been identified only in the South Coast region of KwaZulu-Natal Province. Intense management strategies, including scouting, removal of infected plants, and control of aphids, have been implemented in areas where positive samples were identified to minimize the spread of the virus.

Molecular Variability and Host Distribution of 'Candidatus Phytoplasma solani' Strains from Different Geographic Origins.

The knowledge of phytoplasma genetic variability is a tool to study their epidemiology and to implement an effective monitoring and management of their associated diseases. ‘Candidatus Phytoplasma solani’ is associated with “bois noir” disease in grapevines, and yellowing and decline symptoms in many plant species, causing serious damages during the epidemic outbreaks. The epidemiology of the diseases associated with this phytoplasma is complex and related to numerous factors, such as interactions of the host plant and insect vectors and spreading through infected plant propagation material. The genetic variability of ‘Ca. P. solani’ strains in different host species and in different geographic areas during the last two decades was studied by RFLP analyses coupled with sequencing on vmp1, stamp, and tuf genes. A total of 119 strains were examined, 25 molecular variants were identified, and the variability of the studied genes was linked to both geographic distribution and year of infection. The crucial question in ‘Ca. P. solani’ epidemiology is to trace back the epidemic cycle of the infections. This study presents some relevant features about differential strain distribution useful for disease monitoring and forecasting, illustrating and comparing the phytoplasma molecular variants identified in various regions, host species, and time periods.

Ceratocystis ficicola causing a serious disease of Ficus carica in Greece

Ceratocystis ficicola causes vascular wilt of fig trees in Japan, invading root systems and the main stems eventually leading to tree death. In surveys from 2018 to 2020 in fig orchards in Greece, this fungus was detected in two separated regions. The fungus was consistently isolated from infected wood and from rhizosphere soil. The isolates were identified based on multi-locus phylogenetic analyses of rpb2, bt1 and tef1 gene regions and detailed morphological characteristics, including comparisons with an ex-type isolate of C. ficicola from Japan. The pathogenicity of Greek isolates was proven on Ficus carica and F. benjamina plants. Ceratocystis ficicola is a soil-borne pathogen, and the occurrence of vascular wilt outbreaks suggest that the pathogen spreads within and between orchards with infested soil and wood debris during ploughing. The pathogen is also spreading in Greece with infected propagation material. This is the first detailed report of C. ficicola outside Japan, and there is concern over potential spread of the pathogen to other Mediterranean countries, where approx. 70% of the world fig production occurs.

First report of Tomato spotted wilt virus in Petasites japonicus in Korea.

Butterbur (Petasites japonicus [Siebold & Zucc.] Maxim.) is a perennial herb of the Asteraceae family that is cultivated for medicinal and nutritional purposes. Due to long-term vegetative propagation of virus-infected native species, the yield and quality of butterbur plants have deteriorated. Five viruses have been reported to infect this species: alfalfa mosaic virus (AMV), arabis mosaic virus (ArMV), butterbur mosaic virus (ButMV), broad bean wilt virus 2 (BBWV-2), and cucumber mosaic virus (CMV) (Ham et al. 2016; Tochihara and Tamura 1976). From 2018 to 2019, butterbur plants in four greenhouses in Nonsan, South Korea (Supplementary Figure S1a, b) were found to show virus-like symptoms such as chlorotic and necrotic ring spots, necrosis, and mild mosaic on the leaves. Disease incidence was greater than 80% in one greenhouse (~1,000 m2). To identify the causal virus, we collected 17 symptomatic butterbur leaf samples from these greenhouses and performed reverse-transcription polymerase chain reaction (RT-PCR) analysis using species-specific detection primers for the five reported viruses and tomato spotted wilt virus (TSWV) (Supplementary Table S2). RT-PCR results showed that 12 samples from three greenhouses showing necrotic ring spots and mosaic symptoms were infected with a mixture of TSWV and ButMV, whereas 5 samples from one greenhouse showing mild mosaic symptoms were infected only with ButMV. TSWV (genus Orthotospovirus, family Tospoviridae) is transmitted by thrips and causes serious damage to a wide range of economically important plants (Pappu et al. 2009). ButMV (genus Carlavirus, family Betaflexiviridae) is transmitted by aphids, as well as infected vegetative propagation material (Hashimoto et al. 2009) and is the most predominant virus in butterbur in Korea (Ham et al. 2016). To isolate TSWV from butterbur, leaf extracts from symptomatic samples were mechanically inoculated on an assay host, Chenopodium quinoa, via three single-lesion passages followed by propagation in Nicotiana tabacum cv. Samsun. Thirty different indicator plant species were used for the bioassay of the TSWV isolate (TSWV-NS-BB20) by mechanical inoculation method (Supplementary Table S3). RT-PCR analysis confirmed that TSWV-NS-BB20 induced necrotic local lesions and mosaic on Nicotiana species and ring spots and mosaic on tomatoes and peppers. Notably, TSWV-NS-BB20 reproduced necrotic local lesions and mild mosaic symptoms on butterbur plants which were infected with ButMV with no obvious symptoms. To characterize TSWV-NS-BB20 genetically, the complete genome sequences of L (8914 nt), M (4751 nt), and S (2917 nt) RNA segments were obtained by RT-PCR using specific primers for TSWV as described previously (Kwak et al., 2020). The obtained sequences were deposited in GenBank under accession nos. MT643236, MT842841, and MN854654, respectively. BLASTn analysis showed that sequences of each segment had maximum nucleotide identities of 99.0, 98.9, and 98.6% to TSWV-L, M, and S (KP008128, FM163373, and KP008129) of TSWV-LL-N.05 isolate from tomato in Spain. Since 2018, TSWV outbreaks on butterbur are observed every year and thus may act as a potential source of TSWV infection for other crops of importance to Korea, such as pepper. Owing to the butterbur vegetative propagation, the identification of TSWV infection in butterbur will be helpful for future virus management to generate virus-free materials. To our knowledge, this is the first report of TSWV infection of butterbur.

Virus Pathogens in Australian Vineyards with an Emphasis on Shiraz Disease.

Grapevine viruses are found throughout the viticultural world and have detrimental effects on vine productivity and grape and wine quality. This report provides a comprehensive and up-to-date review on grapevine viruses in Australia with a focus on “Shiraz Disease” (SD) and its two major associated viruses, grapevine virus A (GVA) and grapevine leafroll-associated virus 3 (GLRaV-3). Sensitive grapevine cultivars like Shiraz infected with GVA alone or with a co-infection of a leafroll virus, primarily GLRaV-3, show symptoms of SD leading to significant yield and quality reductions in Australia and in South Africa. Symptom descriptors for SD will be outlined and a phylogenetic tree will be presented indicating the SD-associated isolates of GVA in both countries belong to the same clade. Virus transmission, which occurs through infected propagation material, grafting, and naturally vectored by mealybugs and scale insects, will be discussed. Laboratory and field-based indexing will also be discussed along with management strategies including rogueing and replanting certified stock that decrease the incidence and spread of SD. Finally, we present several cases of SD incidence in South Australian vineyards and their effects on vine productivity. We conclude by offering strategies for virus detection and management that can be adopted by viticulturists. Novel technologies such as high throughput sequencing and remote sensing for virus detection will be outlined.

Grapevine rootstocks can be a source of infection with non-regulated viruses

Grapevine can be infected by several viruses and viroids, the presence of which can lead to yield losses and vineyard decline. Our previous survey of vineyards in Hungary suggested that viral infection originates from infected propagation material. To investigate whether rootstocks can be a source of virus infections, we surveyed seventeen rootstock vineyards and two rootstock collections in Hungary to determine the virome by high-throughput sequencing of small RNAs. The presence of the viruses was also tested by RT-PCR. The results showed that viruses whose presence is routinely checked were almost absent in rootstock vineyards but were present in rootstock genotype collections. Moreover, first the time in Hungary, we detected the presence of Australian grapevine viroid in the rootstock genotype collection at Pecs. In contrast, viruses that are not regulated or not routinely tested, namely, grapevine rupestris stem-pitting-associated virus, grapevine Syrah virus-1 and grapevine Pinot gris virus, were detected in almost all locations in most of the varieties. The presence and absence of infected rootstock genotypes in the same vineyard together with phylogenetic analysis suggested that viral infections originated from infected propagation material. Moreover, we found the symptomatic variant of grapevine Pinot gris virus in several rootstock vineyards without symptoms, suggesting the possibility for leaf mottling and deformation disease symptoms to manifest on susceptible cultivars following grafting onto these rootstocks.

Identification of phytoplasmas and Auchenorryncha in Tunisian vineyards

To verify the risk that plant pathogens such as phytoplasmas could have been introduced with infected propagation material, a preliminary investigation for the presence of phytoplasmas and their insect vectors in the main Tunisian viticultural regions was carried out. To identify phytoplasmas and leafhopper fauna several survey have been carried out on grapevine and spontaneous plant species in the main grapevine growing regions of Tunisia, i.e. Rafraf, Baddar, Mraissa, Belli, Gobba, and Bousalem. A total of 1,109 insect specimens were collected and 30 species were identified, 25 Cicadomorpha and 5 Fulgoromorpha. The 82.58% of the leafhopper species recorded are reported either as vectors of viruses or of phytoplasmas or direct damaging pests, while the 17.49% remaining do not have an important role in field management. In some of the collected grapevine materials aster yellows phytoplasmas (16SrI-B) were identified, confirming previous reports.

Economic Benefits from Virus Screening: A Case Study of Grapevine Leafroll in the North Coast of California

Viruses and related pathogens have no cure and impose high costs on nurseries and specialty crop producers. These diseases are typically spread through infected planting stock and plant propagation material. For some viruses, field spread after planting is also important in determining vineyard health. However, virus spread can be minimized if clean stock is planted. This paper presents the costs and benefits of a virus screening program for Grapevine Leafroll associated Virus-3 (GLRaV-3) in the North Coast region of California. Grower costs and benefits from using GLRaV-3-free vines were computed and extrapolated to the North Coast winegrape industry as a whole. Economic benefits from the GLRaV-3 testing, therapy, and distribution programs were in excess of $20 million/yr for the region and substantially outweighed the costs. The results showed potential benefits from removing and replacing diseased vines, rather than leaving them in the vineyard where they can be foci for disease spread. Significant costs were also associated with disease entering from infected vines on neighboring properties.

IDENTIFICATION OF GRAPEVINE PINOT GRIS VIRUS IN SARDINIA AND LAZIO (SOUTH AND CENTRAL ITALY)

Grapevine Pinot gris virus (GPGV) was identified in several grapevine cultivars, particularly in cv 'Vermentino' showing leaf mottling and deformation, in the Sardinia and Lazio regions of Italy. The majority of GPGV-infected cultivars were asymptomatic but the virus was not detected in samples from vineyards older than 10 years and/or planted with local cultivars, confirming the recent introduction of the virus in these two regions likely through the distribution and use of infected propagation material. Similar to reports from other regions, the high incidence of GPGV in asymptomatic vines is creating challenges with regard to control.

First Report of Banana bunchy top virus in Banana (Musa spp.) from South Africa

Bananas (Musaceae, Zingiberales) are considered to be one of the most important fruit crops and an important staple food in many developing tropical and subtropical regions (Kumar et al., 2011). In Southern Africa, banana production is under threat from a number of viral diseases, including Banana bunchy top virus (BBTV) (family Nanoviridae, genus Babuvirus). BBTV is the cause of Banana bunchy top disease, the most devastating virus disease of banana. In Sub-Saharan Africa the disease is endemic to 13 countries, including Malawi and Zambia in the Southern African sub-region. Previously, a detection survey for BBTV was done in South Africa in 1996 and this study confirmed the absence of BBTV in the Kiepersol region of Mpumalanga Province (Pietersen et al., 1996). No other surveys were conducted since then in South Africa. The disease is spread by the banana aphid Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae), that is present in South Africa, and through infected propagation material. Pentalonia nigronervosa feeds on the phloem of host plants, and potentially picks up the BBTV particles from the phloem and spreads them in plantations as they feed. In June 2015, five 'Williams' plants, showing symptoms resembling BBTV, including streaks on the pseudostem and upright bunchy appearance, were submitted for identification to the Virology Diagnostic Lab at Agricultural Research Council-Plant Protection Research Institute (ARC-PPRI). The plants originated from a farm close to Hibberdene in the KwaZulu Natal South Coast production region (GPS coordinates: S 300 30, 636', E 300 30,653'). A BBTV-specific PCR was done using the protocol published by Thomson and Dietzgen (1995), using primer pairs BBT-1 and BBT-2, amplifying a fragment of the putative replicase gene. The five plants tested positive for BBTV, and the PCR products (~349 bp) of two of the accessions, 15/1408 and 15/1410, were directly sequenced using Sanger sequencing and 321 bp of both sequences were deposited in GenBank (GenBank Accession Nos: KU196167, KU196168). A non-infected banana plant was included in the test as a healthy control. Phylogenetic analyses of the partial replicase genomic region grouped accessions 15/1408 and 15/1410 with the South Pacific group that included accessions from India, Pakistan and other regions in Africa with a sequence identity of 99% (Kumar et al., 2011). A second confirmation test for the presence of BBTV in these plants was conducted using a BBTV-specific Enzyme-linked immunosorbent assay (ELISA) supplied by Agdia (Catalog number: SRA24700) using the manufacturer's instructions. The five plants tested positive for BBTV in the ELISA test, confirming the previous positive results. Forty-four additional samples were included in the ELISA test. These samples were collected randomly from 14 blocks on the same farm (GPS coordinates: S 300 30, 636', E 300 30,653') and comprised of symptomatic and non-symptomatic plants. BBTV was detected with ELISA in 34 of these plants, ten plants tested negative for BBTV. In one of these blocks, C17, all the sampled plants (4), tested negative. In three other blocks (A9, C8 and C15), positive and negative plants were recorded. In the remaining 10 blocks, all collected plants tested positive. Intense management strategies, including removal of infected plants and spraying for aphids, were implemented on the farm to prohibit the spread of the virus to neighbouring plantations. The ELISA test confirmed the earlier detection of BBTV with PCR and sequencing. This is the first report of BBTV on Musa spp. from South Africa. Strict implementation of phytosanitary measures will be necessary to prevent the spread of this virus to other regions in the country. The need to do extensive surveys for the presence of BBTV in other banana production regions in South Africa is highlighted by this finding.

Visualization of ‘Candidatus Liberibacter asiaticus’ Cells in Citrus Seed Coats with Fluorescence In Situ Hybridization and Transmission Electron Microscopy

‘Candidatus Liberibacter asiaticus’ is bacterium implicated as the causal agent of the economically damaging disease of citrus called huanglongbing (HLB).  The bacterium is spread by movement of infected citrus propagation material and by the Asian citrus psyllid, Diaphorina citri Kuwayama.  Seed transmission is a possible additional route of dissemination for the pathogen.  Some published studies on seed transmission found abundant bacterial DNA in seed coats but no infections of germinated seedlings.  No direct observations of bacteria in seed coats were made in these studies.  In vascular bundles from citrus seed coats, we confirmed the presence of bacterial cells with transmission electron microscopy (TEM) and Fluorescence In Situ Hybridization (FISH).  The physical measurements and the morphology of individual bacterial cells revealed by TEM and FISH were consistent with those described in the literature for ‘Ca. Liberibacter asiaticus’.  No bacterial cells were observed in seed coats from non-infected trees.  A library of clones of prokaryote 16S rRNA gene sequences amplified from a non-infected tree contained no ‘Ca. Liberibacter asiaticus’ sequences, whereas 95% of the sequences in a similar library prepared from an infected tree were ‘Ca. Liberibacter asiaticus’, providing additional data that the bacterial cells observed by TEM and FISH in seed coats from infected trees were ‘Ca. Liberibacter asiaticus’.

Ideas on the European stone fruit yellows – as an entomologist can see them

The European stone fruit yellows (ESFY) is an important endemic disease in Europe which causes in both, the Mediterranean countries and Central Europe serious damage. Its pathogen is the ‘Candidatus Phytoplasma prunorum’. The treatment and healing of the diseased trees and plantations with chemicals do not promise success. Thus, prevention may be the only solution. The transmission and spread of the pathogen happen by infected propagation material (grafting) or a vector (the psyllid, Cacopsylla pruni). Mechanism of the pathogen’s transmission and population dynamics of the vector have been extensively investigated in several European countries, which may allow by the control of C. pruni even to hold back the disease. Diseased stone fruit trees and wild Prunus spp. as main host species play an important role in maintaining and spreading the pathogen. C. pruni collects the pathogen by feeding on these plants and it carries persistently ‘Ca. P prunorum’. Researchers in Hungary have been characterized the disease only in terms of plant pathology, but neither the significance of the vector nor the role of wild Prunus spp. have been studied. This summary intends to give clues to these researches, that not only axe and saw should be the instruments of national control, but knowing the role and population dynamics of the vector the stone fruit production should be more successful.

Variability and genetic structure of a natural population of Citrus psorosis virus

In this study, we examined the population structure and genetic diversity of Citrus psorosis virus (CPsV) in Morocco. Analysis of the coat protein partial sequences of 34 isolates collected in the three main citrus-growing areas of Morocco showed that CPsV grouped in three major groups, with low within-group nucleotide diversity. Analyses indicated that CPsV genetic diversity is not structured by the geographic origin of the CPsV isolates. The genetic variation resulting from mutations depends on evolutionary forces that have contributed to the shaping of the genetic structure and diversity of the CPsV populations analyzed here: negative selective pressure for amino acid variation, recombination between variants or mixed infection, genetic drift induced by the founder effect associated with the transmission process, and migration explained by the exchange of infected propagative plant material.

Detection of a new variant of Citrus tristeza virus in Greek citrus crops

Citrus tristeza virus (CTV), the most destructive virus of citrus, is a quarantine pathogen in Greece. Since 2000, several accidental imports of infected propagation material have been detected in the country, and while eradication measures were applied, a few disease foci still remain. CTV isolates were collected from Chania (Crete) and the “lemonwood” of Poros (Peloponnese), and their genetic variability was studied using single-strand conformation polymorphism (SSCP). One previously characterized isolate from Argolida grafted on a Mexican lime (GR3) and two Italian isolates from Calamondin were also included in the study. ELISA and RT-PCR tests confirmed CTV presence, and SSCP analysis of the virus amplified coat protein (CP) gene was used to separate either distinct virus isolates for cloning the CP gene or variants (haplotypes) for sequencing. Analyses showed that selected variants of four representative isolates clustered into three of the seven defined phylogenetic groups: groups 3b and 5 (severe isolates) and group M (mild isolates). The prevalent haplotypes detected in the CTV from lemonwood of Poros (GR9) were in group 3b, confirming previous results. However, one sequence variant was identified as a recombinant between haplotypes from groups 3b and 5. Variants of these two groups were also detected in the Italian Calamondin isolate. In the grafted Mexican lime isolate (GR3) from Argolida, only one haplotype was found which belonged to group M, while in the field isolate from Chania (GR6) the only haplotype detected was in group 5. This is the first report of variants of group 5 in Greece, suggesting an unknown virus introduction. The prevalence of severe isolates in the area is of particular concern, and implications for the future of the CTV epidemics are discussed.

Infection of Sweetpotato by Fusarium solani and Macrophomina phaseolina Prior to Harvest.

The end rot disease complex, caused mainly by Fusarium solani and Macrophomina phaseolina, can be an important postharvest problem in sweetpotato. The disease develops a few weeks after storage roots are harvested and stored. Isolations attempted after harvest showed that the pathogens can be present inside the storage roots before symptoms appear. To determine how and when end rot pathogens enter sweetpotato storage roots, two greenhouse experiments were designed using tissue culture-derived plants free of F. solani and M. phaseolina. In one experiment, plants were grown in autoclaved soil, and 1 month after transplanting, plants were inoculated at the soil line with either noninfested toothpicks or toothpicks infested with each fungus alone or combined. In the other experiment, plants were grown in noninfested soil or in soil infested with each fungus alone or combined. F. solani and M. phaseolina were isolated from roots, storage roots, and plant stems below the soil line, at the soil line, and 5 cm above the soil line in both experiments. This suggests these fungi are capable of invading sweetpotato plants and storage roots from infested soil, and can systemically colonize the plant from infected plant propagation material.

First Report of Xanthomonas citri pv. citri-A Causing Asiatic Citrus Canker in Mayotte.

Asiatic citrus canker, caused by Xanthomonas citri pv. citri, is a bacterial disease of major economic importance in tropical and subtropical citrus-producing areas. X. citri pv. citri pathotype A can cause severe infection in a wide range of citrus species and induces erumpent, callus-like lesions with water-soaked margins evolving to corky cankers and leading to premature fruit and leaf drop and twig dieback on susceptible/very susceptible cultivars. A chlorotic halo is typically visible around canker lesions on leaves and young fruit, but not on mature fruit and twigs. This quarantine organism can strongly impact both national and international citrus markets. Long distance dispersal is mainly through infected propagative material. Asiatic citrus canker occurs on most islands in the Southwest Indian Ocean region (Comoros, Mauritius, Reunion, Rodrigues, and Seychelles islands), but was not yet reported in Mayotte (EPPO-PQR available at http://www.eppo.int ). In May 2012, typical canker-like symptoms were observed on sweet orange (Citrus sinensis) groves on Mtsamboro islet and soon after on the main island of Mayotte, mostly on sweet oranges, but also on Tahiti limes (C. latifolia) and mandarins (C. reticulata). Eighty-one Xanthomonas-like strains were isolated using KC semi-selective medium (4) from disease samples collected from both commercial groves and nurseries on different Citrus species located all over the island. Sixteen Xanthomonas-like isolates were tentatively identified as X. citri pv. citri based on a specific PCR assay with 4/7 primers (3). All strains but the negative control, sterile water, produced an amplicon of the expected size similar to X. citri pv. citri strain IAPAR 306 used as positive control. Multilocus sequence analysis targeting six housekeeping genes (atpD, dnaK, efp, gltA, gyrB, and lepA) (1,2) fully identified three strains from Mayotte (LJ225-3, LJ228-1, and LJ229-11) as X. citri pv. citri (and not other xanthomonad pathovars pathogenic to citrus or host range-restricted pathotypes of pathovar citri), and more specifically as sequence type ST2 composed of pathotype A strains of X. citri pv. citri (2) (including all strains from the Southwest Indian Ocean region). Eight strains were inoculated by a detached leaf assay (2) to Mexican lime SRA 140 (C. aurantifolia), Tahiti lime SRA 58, sweet orange cv. Washington Navel, alemow SRA 779 (C. macrophylla), and tangor cv. Ortanique (C. reticulata × C. sinensis) and developed typical erumpent, callus-like tissue at wound sites for all Citrus species, fulfilling Koch's postulates. Xanthomonas-like yellow colonies were reisolated from symptoms produced by the eight strains inoculated on Mexican lime. Boiled bacterial suspensions were assayed by PCR with 4/7 primers (3) and produced the expected 468-bp amplicon in contrast with the negative control (sterile water). No lesions developed on the negative control consisting of inoculations by 10 mM tris buffer (pH 7.2). Citrus canker-free nurseries and grove sanitation should be implemented for decreasing the prevalence of Asiatic canker in this island territory.

First Report of Sequence Type 1, Pathotype A Xanthomonas citri pv. citri from Lime and Lemon Fruit Originating from Bangladesh.

Asiatic canker caused by Xanthomonas citri pv. citri, a quarantine pest in several countries (including the European Union), strongly impacts both national citrus markets in tropical and subtropical areas and international trade. This bacterium induces erumpent, callus-like lesions often with a water-soaked margin in a wide range of citrus species causing premature fruit drop and twig dieback. Long distance dispersal is mainly through infected propagative material and the role of fruit is still debated. During inspection of imported limes (C. aurantifolia) and lemons (C. limon) from Bangladesh from 2006 to 2009, canker-like infected fruits were intercepted by the UK plant health service. Typical corky lesions were surface sterilized and comminuted in 0.1% peptone solution. Suspensions were plated onto nutrient dextrose (ND) and yeast dextrose chalk (YDC) plates for bacterial isolation. After incubation for 3 to 7 days at 25°C, typical Xanthomonas-like yellow colonies were purified for identification. Identification of 18 isolates as Xanthomonas was carried out initially by fatty acid methyl ester (FAME) analysis. Identification at the species level (X. citri) was completed by sequencing of the gyrase B gene (4). PCR (3) was used to confirm the identity of these isolates using X. citri pv. citri CFBP 2525 as the positive control and distilled water as the negative control. The expected DNA fragment was only obtained from all of the bacterial isolates using primer pair 4/7 (3). Multilocus sequence analysis (MLSA) of four housekeeping genes (atpD, dnaK, efp, and gyrB) identified isolates from Bangladesh as two sequence types of X. citri pv. citri, ST1 (n = 5; GenBank Accession Nos. FJ376118, FJ376168, FJ376216, and FJ376251) and ST2 (n = 13; EU333904, EU333907, EU333910, and FJ376357), but not as any other xanthomonad pathogenic to citrus (2). Amplified fragment length polymorphism (AFLP) analysis of all X. citri pv. citri isolates from Bangladesh and additional reference isolates from pathotype A, A*, Aw and X. citri pv. aurantifolii (2) using Sac I/Msp I and four primer pairs (unlabelled MspI + 1 (A, C, T, or G) primers and 5'-labeled - SacI + C primer for the selective amplification step) confirmed identification as X. citri pv. citri. All five ST1 isolates grouped as a single cluster by AFLP, although not strongly supported by bootstrap analysis. Evolutionary genome divergences (EGD) computed from AFLP data ranged from 0.0000 to 0.0097 (median EGD 0.0055) suggested a relatively wide diversity within isolates originating from Bangladesh (median EGD from a worldwide pathotype A collection [n = 73] 0.0028) (2). When inoculated to Mexican lime SRA 140 and grapefruit cv. Duncan using a detached leaf assay (2), all the Bangladesh isolates produced typical extensive canker lesions on both species whereas the negative control (10 mM Tris buffer pH 7.2) did not, and Koch's postulates were fulfilled. To our knowledge, this is the first report of pathotype A assigned to ST1 by MLSA. All strains previously assigned to ST1 displayed a narrow host range (pathotype A*) (2). Our results further identify the Indian subcontinent as an area of relatively wide genetic diversity of X. citri pv. citri (1).