Colonization of citrus seed coats by 'Candidatus Liberibacter asiaticus': implications for seed transmission of the bacterium

Huanglongbing is an economically damaging disease of citrus associated with infection by 'Candidatus Liberibacter asiaticus'. Transmission of the organism via infection of seeds has not been demonstrated but is a concern since some citrus varieties, particularly those used as rootstocks in commercial plantings are propagated from seed. We compared the incidence of detection of 'Ca. Liberibacter asiaticus' DNA in individual fruit peduncles, seed coats, seeds, and in germinated seedlings from 'Sanguenelli' sweet orange and 'Conners' grapefruit fruits sampled from infected trees. Using real-time quantitative PCR (qPCR) we detected pathogen DNA in nucleic acid extracts of 36 and 100% of peduncles from 'Sanguenelli' and from 'Conners' fruits, respectively. We also detected pathogen DNA in extracts of 37 and 98% of seed coats and in 1.6 and 4% of extracts from the corresponding seeds of 'Sanguenelli' and 'Conners', respectively. Small amounts of pathogen DNA were detected in 10% of 'Sanguenelli' seedlings grown in the greenhouse, but in none of 204 extracts from 'Conners' seedlings. Pathogen DNA was detected in 4.9% and in 89% of seed coats peeled from seeds of 'Sanguenelli' and 'Conners' which were germinated on agar, and in 5% of 'Sanguenelli' but in none of 164 'Conners' seedlings which grew from these seeds on agar. No pathogen DNA was detected in 'Ridge Pineapple' tissue at 3 months post-grafting onto 'Sanguenelli' seedlings, even when pathogen DNA had been detected initially in the 'Sanguenelli' seedling. Though the apparent colonization of 'Conners' seeds was more extensive and nearly uniform compared with 'Sanguenelli' seeds, no pathogen DNA was detected in 'Conners' seedlings grown from these seeds. For either variety, no association was established between the presence of pathogen DNA in fruit peduncles and seed coats and in seedlings.     

The question of seed transmissibility of Plum pox virus

For many years, Plum pox virus (PPV) was considered to be transmissible by seed, increasing the fear of long-distance spread of the disease. In the late 1970s, it was claimed on the basis of biological transmission of the virus to herbaceous indicator plants and the development of serological diagnosis based on polyclonal antibodies, that PPV was seed-transmitted, with a different infection rate according to the plant species and part of the seed which was tested. In the 1990s, PPV was characterized into four different types, and specific monoclonal antibodies were produced for them. These new and more sensitive diagnostic techniques, together with RT-PCR with different sets of specific primers, were used to approach once again the problem of PPV transmission through seeds. The virus was detected in seed coats and cotyledons, but embryonic tissue and seedlings obtained from germinated seeds never showed symptoms, and gave negative results for PPV with both ELISA and PCR assays. No PPV isolate is currently recognized to be seed transmitted, so vertical transmission of PPV from infected mother plants to their progeny does not occur. Hypothetically, the only possibility of seed transmission would arise from a mutation in the helper component of the virus, associated with high susceptibility of the infected Prunus cultivar.     

Tracing seed to seedling transmission of the wheat blast pathogen Magnaporthe oryzae pathotype Triticum

Wheat blast caused by Magnaporthe oryzae pathotype Triticum (MoT), initially restricted to South America, is a global threat for wheat after spreading to Asia in 2016 by the introduction of contaminated seeds, raising the question about transmission of the pathogen from seeds to seedlings, a process so far not well understood. We therefore studied the relationship between seed infection and disease symptoms on seedlings and adult plants. To accomplish this objective, we inoculated spikes of wheat cv. Apogee with a transgenic isolate (MoT-DsRed, with the addition of being resistant to hygromycin). We identified MoT-DsRed in experiments using hygromycin resistance for selection or by observation of DsRed fluorescence. The seeds from infected plants looked either apparently healthy or shrivelled. To evaluate the transmission, two experimental designs were chosen (blotter test and greenhouse) and MoT-DsRed was recovered from both. This revealed that MoT is able to colonize wheat seedlings from infected seeds under the ground. The favourable conditions of temperature and humidity allowed a high recovery rate of MoT from wheat shoots when grown in artificial media. Around 42 days after germination of infected seeds, MoT-DsRed could not be reisolated, indicating that fungal progression, at this time point, did not proceed systemically/endophytically. We hypothesize that spike infection might occur via spore dispersal from infected leaves rather than within the plant. Because MoT-DsRed was not only successfully reisolated from seed coats and germinating seeds with symptoms, but also from apparently healthy seeds, urgent attention is needed to minimize the risks of inadvertent dispersal of inoculum.     

Orchid fleck symptoms may be caused naturally by two different viruses transmitted by <Emphasis Type="Italic">Brevipalpus</Emphasis>

Brevipalpus-transmitted viruses (BTV) cause chlorotic, necrotic and/or ringspot lesions in leaves and stems of orchids, citrus, coffee and several other plant species. There are two different types of BTVs, the nuclear and the cytoplasmic, based on maturation locale in the cell and particle morphology. The orchid fleck virus (OFV) is a BTV that infects orchids. Its short rodlike particles are 32–40 nm in diameter, 100–150 nm in length. OFV is found in the nucleus and is associated with intranuclear electronlucent viroplasms. In 1999, transmission electron microscopy analysis revealed a distinct type of virus causing orchid fleck symptoms. The bacilliform particles, 70–80 nm in diameter and 110–120 nm in length, induced electron-dense viroplasm inclusions in infected cells and resembled the cytoplasmic type associated with BTV, such as the citrus leprosis virus C. Our objective in the present study was to verify whether the cytoplasmic type virus found in orchids could be amplified using primers for other cytoplasmic BTVs, such as CiLV-C and Solanum violaefolium ringspot virus (SvRSV). Additionally, we aimed to differentiate the two BTVs found in orchids: the nuclear and the cytoplasmic types of OFV using microscopy and molecular and serological tools. This virus was not amplified by the CiLV-C and SvRSV primers, and neither the molecular nor the serological tools available to the OFV diagnosis reacted with it, demonstrating that they are definitely different viruses.     

Zucchini yellow mosaic virus; two outbreaks in the Netherlands and seed transmissibility

In 1983 and 1987/88 two limited outbreaks of zucchini yellow mosaic virus in cucumber and zucchini squash occurred in the Westland glasshouse district in the Netherlands, mainly in glasshouses. The disease could be eradicated and has not recurred so far. In both cases a relatively mild but still highly pathogenic strain of the virus was involved. Diseased plants of zucchini yielded severely distorted or no fruits and it was difficult to obtain seeds from infected plants. Two out of 4196 seedlings grown in isolation from seed from inoculated zucchini plants showed symptoms and contained the virus, indicating that the virus can be transmitted via seed but at very low rate. This explains the erratic incidence and international distribution of the virus.     

Spatial distribution of purple seed stain of soybean caused by <Emphasis Type="Italic">Cercospora kikuchii</Emphasis> in fields

To investigate the frequency distribution of purple seed stain of soybean caused by Cercospora kikuchii in two experimental fields in 2004, we set up rows 75 cm apart and sowed two asymptomatic seeds at each of positions 20 cm apart in each row. We sowed purple-stained seeds infected with the pathogen as inocula at four points instead of asymptomatic seeds in each field. We assessed disease incidence in harvested seeds by counting the numbers of purple-stained and asymptomatic seeds. To determine the spatial distribution of the disease, we grouped the field points into analytical units of several sizes. Beta-binomial and binomial distributions described the distribution patterns of purple-stained seeds. The smallest value of α, a beta-binomial parameter, occurred with analytical units that contained three or nine points next to each other within a single row, suggesting that these units showed the most aggregated distribution of the disease, each of the patches of seeds infected with C. kikuchii could be defined approximately by the area covered by three or nine points (75 × 60 or 75 × 180 cm), and the disease tended to infect plants next to each other within rows.     

Seed and pollen transmission of <Emphasis Type="Italic">Apple latent spherical virus</Emphasis> in apple

To examine whether Apple latent spherical virus (ALSV) has spread among apple trees in an orchard, we surveyed 21 apple trees surrounding two ALSV-infected trees for virus infection using a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). None of the 21 trees were infected, indicating that ALSV has not spread from the infected trees to the neighboring apple trees since it was first detected in 1984. We analyzed seed embryos and seedlings derived from infected trees and detected ALSV in 10 of 223 seed embryos (4.5%) and 10 of 227 seedlings (4.4%). From these results, we conclude that ALSV is seed-transmitted at a rate of ca. 4.5% in apple. We also analyzed seed embryos and seedlings from uninfected apple trees that were hand-pollinated with pollen from infected trees. We detected ALSV in only 1 of 260 seed embryos and in none of the 227 apple seedlings. This result indicated that the seed transmission rate via infected pollen is only 0–0.38%. In situ hybridization analysis of ALSV-infected apple flower buds showed that ALSV was present inside almost all pollen grains and in all ovary and ovule tissues, including the embryo sac and inner integument.     

High incidence of seed transmission of Papaya ringspot virus and Watermelon mosaic virus, two viruses newly identified in Robinia pseudoacacia

Black locust (Robinia pseudoacacia L.) is an ornamental legume tree susceptible to several viruses. Virus diseases of black locust often go unrecognized making infected trees a prime inoculum source, not only for legumes, but also other plant species. In this communication we report, for the first time, two viruses, Papaya ringspot virus and Watermelon mosaic virus, associated with disease in black locust. Isolates of both viruses were partially characterized and seed transmission was examined. The high percentage of seed transmission suggests that this may be an important avenue for virus dissemination.     

Seed transmission of Melon necrotic spot virus and efficacy of seed-disinfection treatments

Rates of seed transmission of Melon necrotic spot virus (MNSV) were estimated in seedlings grown from commercial melon ( Cucumis melo ) cv. Galia F₁ seeds. Seedlings at the cotyledon stage and adult plants were assayed for MNSV by DAS-ELISA and RT-PCR. None of the seedling groups tested positive for MNSV by ELISA. The proportion of seedlings infected with MNSV was at least 7 and 8% in seed lots 05 and 06, respectively, as estimated from RT-PCR analysis of grouped seedlings. Fourteen and eight grouped samples (10 seedlings per group), of a total of 200 and 100 seedlings, respectively, grown from infected seeds were MNSV-positive in seed lots 05 and 06, respectively, corresponding to seed-to-seedling transmission rates of 11·3 and 14·8%, respectively. Several seed-disinfection treatments were evaluated for their ability to prevent seed transmission of MNSV. The results suggest that a treatment of 144 h at 70°C can be used to eradicate MNSV in melon seeds without hindering germination.     

Alfalfa mosaic virus in lucerne seed during seed maturation and storage, and in seedlings

The incidence of alfalfa mosaic virus (AMV) in lucerne seed and pods during maturation, when monitored by sap transmission to Phaseolus (infective virus) and ELISA (AMV antigen), showed that infective virus incidence decreased rapidly with maturation, whereas antigen incidence declined slowly and was always higher than infective virus. Infective virus and antigen incidence were higher in mature seed of cv. Maris Kabul than cv. Europe because virus inactivation/degradation were more rapid in cv. Europe. Seed infection with virus originating from pollen, ovules or both was found in pods and seeds 12–15 days after pollination between healthy or AMV-infected plants; this was before maturation-associated virus inactivation. Ovule transmission was more frequent than pollen transmission. AMV antigen was present in embryos and testas of mature seed; infective virus only in embryos. Non-infective but ELISA-positive antigen in testa extracts accounted for the higher incidence of 'seed-borne AMV' compared with embryo-associated seed transmission to seedlings. Tests with dry mature seed either underestimated (infectivity tests) or overestimated (ELISA) eventual seedling infection. Infectivity and ELISA tests gave identical incidence values for 17 to 29-day-old seedlings.     

Alternative hosts and seed transmissibility of soybean blotchy mosaic virus

Soybean blotchy mosaic virus (SbBMV) is an important virus of soybean in the warmer regions of South Africa. The presence of the virus is associated with blotchy mosaic symptoms on soybean leaves and significant annual yield losses. The virus is a member of the genus Cytorhabdovirus and persists between soybean growing seasons. In this study, multiple specimens of indigenous tree species, other crops and herbaceous weeds surrounding soybean fields with high disease incidences of SbBMV were tested for the presence of SbBMV by RT-PCR in order to determine whether the presence of alternative hosts facilitates the seasonal carry-over of the virus. Commercial soybean cultivars commonly grown in the region were also evaluated for seed transmissibility of the virus. A total of 487 accessions representing 27 different species were screened and one accession each of Flaveria bidentis, Lamium amplexicaule and Gymnosporia buxifolia tested positive for the presence of SbBMV and may serve as possible alternative hosts of SbBMV, allowing over-wintering of the virus when soybean is absent. Symptoms associated with SbBMV infection were not present in any of the 2, 829 seedlings collected from naturally infected SbBMV plants, and none of the 21 seedlings showing various abnormalities and tested by RT-PCR were positive. SbBMV does not appear to be seed transmissible in soybean at an incidence above that which numbers screened would have detected the virus. It was concluded that the presence of alternative plant hosts, functioning as viral reservoirs during the soybean off-season might allow for the re-emergence of the disease early in the soybean production season each year. Future work will investigate the role of Peragallia caboverdensis, the leafhopper vector of SbBMV, and specifically the possible propagative transmission of the virus in the persistence of the disease.     

Seed transmission of Sweet potato leaf curl virus in sweet potato (Ipomoea batatas)

Sweet potato leaf curl virus (SPLCV) infects sweet potato and is a member of the family Geminiviridae (genus Begomovirus). SPLCV transmission occurs from plant to plant mostly via vegetative propagation as well as by the insect vector Bemisia tabaci. When sweet potato seeds were planted and cultivated in a whitefly‐free greenhouse, some sweet potato plants started to show SPLCV‐specific symptoms. SPLCV was detected by PCR from all leaves and floral tissues that showed leaf curl disease symptoms. More than 70% of the seeds harvested from SPLCV‐infected sweet potato plants tested positive for SPLCV. SPLCV was also identified from dissected endosperm and embryos. The transmission level of SPLCV from seeds to seedlings was up to 15%. Southern blot hybridization showed SPLCV‐specific single‐ and double‐stranded DNAs in seedlings germinated from SPLCV‐infected seeds. Taken altogether, the results show that SPLCV in plants of the tested sweet potato cultivars can be transmitted via seeds and SPLCV DNA can replicate in developing seedlings. This is the first seed transmission report of SPLCV in sweet potato plants and also, to the authors' knowledge, the first report of seed transmission for any geminivirus.     

Infection process of Stagonosporopsis tanaceti in pyrethrum seed and seedlings

Ray blight disease of pyrethrum (Tanacetum cinerariifolium) is caused by Stagonosporopsis tanaceti, with infected seed being a major means of transmission of this fungal pathogen. The infection process of S. tanaceti in pyrethrum seed and seedlings was determined. Infection hyphae only infected the outer and inner layers of the seed coat and not the embryo of naturally infected pyrethrum seed. During the process of germination of infected seed, S. tanaceti from the seed coat infected the developing embryo and cotyledon, resulting in pre‐ and post‐emergence death, depending on the level of infection in the seed coat. Pre‐emergence death occurred due to disintegration of the infected embryo, which was replaced by hyphae and extracellular anthocyanin‐like material (EAM) at 7 days after incubation (dai). Post‐emergence death occurred after both epidermal and cortical tissues of infected cotyledons at the crown/hypocotyl region disintegrated due to colonization by hyphae. Moreover, most of the tissues of the vascular bundles and cortical tissues contained heavy depositions of EAM at 10–14 dai. In 6‐week‐old infected seedlings, hyphae were confined to the epidermis and the cortical tissues at the crown/hypocotyl regions; the vascular bundles of both infected and uninfected regions, and cortical tissues of the uninfected regions of the seedlings were completely free from infection hyphae and EAM. These findings provide a better understanding of the early stages of the disease cycle of S. tanaceti and will lead to improved control measures for seedborne infection using seed treatments.     

<Emphasis Type="Italic">Erwinia aphidicola</Emphasis> isolated from commercial bean seeds (<Emphasis Type="Italic">Phaseolus vulgaris)</Emphasis>

In late 2003, a new disease appeared in protected bean crops in southeastern Spain, causing a decrease of over 50% in production. Several samples of affected plants were collected and analyzed and the agent of this disease was identified as the bacterium Erwinia aphidicola, which had never been described as a pathogen previously. We attempted to determine the possible bacterium transmission through seeds, using 120 commercial bean seeds from the same batch as that used in an affected farm, and 120 seeds from the fruiting plants of the same farm. Seed coats, cotyledons and leaves of plants originating from them, were taken and analyzed. Several of the developed symptoms on plants from commercial and fruiting plant seeds were internervial chlorosis, necrotic pits and rough roots and they coincided with those observed on affected crops. Bacteria present in commercial seed cotyledons were isolated and analyzed by biochemical and molecular tests. Results confirmed the presence of Erwinia aphidicola in four analyzed seeds; moreover, Bacillus simplex/Bacillus muralis, Pseudomonas mendocina, Pseudomonas putida and Paenibacillus polymyxa were also identified.     

The importance of the infected seed tuber and soil inoculum in transmitting Potato mop‐top virus to potato plants

Potato mop‐top virus (PMTV), the cause of spraing in potato tubers, is transmitted by Spongospora subterranea, the cause of powdery scab, and by planting infected seed tubers. This study was undertaken to determine the relative importance of these sources of infection in seed potato production in Scotland. The transmission of PMTV from tested seed tubers to daughter plants was examined over 2 years and six cultivars. The development of foliar symptoms varied with year and cultivar. Infection of daughter tubers derived from PMTV‐infected seed tubers was more prevalent on plants affected by foliar symptoms than those without symptoms. The rate of transmission of PMTV from infected seed tubers to daughter tubers ranged from 18 to 54%. Transmission was affected by cultivar and by origin of seed tubers used for a cultivar, but not by a cultivar's sensitivity to PMTV infection. The incidence of PMTV in daughter tubers of cv. Cara grown from seed potatoes from one source (common origin) by more than 25 seed producers was examined over two successive generations. The incidence of PMTV in daughter tubers was not correlated with that in the seed tubers but appeared to be strongly associated with soil inoculum. The incidence of PMTV was correlated with powdery scab in those crops in which both were present. There was some evidence from soil tests conducted in 2006 using a tomato bait plant and real‐time RT‐PCR that planting PMTV‐infected seed potatoes could increase the risk of introducing the virus into land not infested by PMTV.     

Endophytes of <Emphasis Type="Italic">Lippia citriodora</Emphasis> (Syn. <Emphasis Type="Italic">Aloysia triphylla</Emphasis>) enhance its growth and antioxidant activity

Sweet pepper (Capsicum annuum) is a popular crop worldwide and an asymptomatic host of the begomovirus (Geminiviridae) Tomato yellow leaf curl virus (TYLCV). A previous study showed that TYLCV could be transmitted by the seeds of tomato plants, but this phenomenon has not been confirmed in other plants. In 2015, four different cultivars of sweet pepper (‘Super Yellow,’ ‘Super Red,’ ‘Sunnyez’ and ‘Cupra’) known to be susceptible to TYLCV were agro-inoculated with a TYLCV infectious clone. Three months after inoculation, the leaves of the ‘Super Yellow’ cultivar showed 80% (8/10) susceptibility and the other three sweet pepper cultivars showed 30 to 50% susceptibilities. All of the ‘Super Yellow’ seed bunches (five seeds per bunch) from plants whose leaves were confirmed to be TYLCV-infected were also TYLCV-infected (8/8). The seeds of other cultivars showed 20 to 40% susceptibilities. Virus transmission rates were also verified with 10 bunches of seedlings for each cultivar (five seedlings per pool). Eight bunches of ‘Super Yellow’ seedlings (8/10) were confirmed to be TYLCV-infected and one to three bunches of each of the other cultivar seedlings were also infected. Viral replication in TYLCV-infected seeds and seedlings was confirmed via strand-specific amplification using virion-sense- and complementary-sense-specific primer sets. This is the first report of TYLCV seed transmission in sweet pepper plants and among non-tomato plants. Because sweet pepper is an asymptomatic host of TYLCV, seeds infected with TYLCV could act as a silent invader of tomatoes and other crops.