Epidemiological aspects of mango malformation disease caused by Fusarium mangiferae and source of infection in seedlings cultivated in orchards in Egypt

Mango malformation, caused by the fungus Fusarium mangiferae , is one of the major diseases of this crop occurring worldwide. This study was conducted to investigate aspects of the epidemiology, survival and spread of the pathogen in general and specifically in seedlings, the majority of which are cultivated in infected orchards in Egypt. Survival of conidia of a representative isolate (506/2) declined very rapidly in soil under summer conditions (1·6 weeks for 50% population decline), but significantly less in controlled and winter conditions (17·9 and 15·0 weeks, respectively, for 50% population decline). Likewise, inoculum survival in naturally infected panicles on the soil surface declined faster than in those buried at 30-cm depths. Natural infections were evaluated on fruits and seeds in a heavily infected and a healthy orchard. In infected trees, the skins of all sampled fruits within a 2-m radius of infected panicles were infected, but the pathogen was not detected in the seeds, seed coats or flesh. The pathogen was not detected in any parts of fruits from a healthy orchard. Vegetatively malformed mango seedlings, growing under infected trees bearing infected panicles, were sampled in two locations in Egypt to determine whether infection in seedlings was systemic (evenly distributed within plant tissue) or whether the pathogen originated from malformed panicles. According to PCR-specific primer amplification, the pathogen was detected in 97% of seedling apical meristems, declining gradually to 5% colonization in roots. It was concluded that inoculum of the pathogen originates from infected panicles and affects seedlings from the meristem, with infections descending to lower stem sections and roots. Minor infections of roots may occur from inoculum originating from infected panicles, but the pathogen is not seedborne.     

<Emphasis Type="Italic">Indian citrus ringspot virus:</Emphasis> localization of virus in seed tissues and evidence for lack of seed transmission

Indian citrus ringspot disease is an important viral disease in kinnow mandarin orchards where disease incidence up to 100% has been recorded. The disease is caused by Indian citrus ringspot virus (ICRSV), a positive sense flexuous RNA virus. The transmission of ICRSV is generally through budwood. Association of ICRSV with pollens of naturally infected flowers from cv. ‘Kinnow’ mandarins has been shown previously and this study demonstrates the presence of ICRSV in seed tissues. DAC-ELISA revealed the presence of virus in seed coats but not in embryo and endosperm of seeds collected from the fruits of ICRSV-infected Kinnow plants. Of the infected seed coats, 18% were found to harbor the virus. The seedlings in the grow-out test did not show any symptom for 2 years and the virus could not be detected in seedlings by DAC-ELISA and RT-PCR. The present study indicated that ICRSV could be localized in the testa of seeds but its transmission to progeny was not observed.     

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.     

苹果锈果类病毒在八棱海棠种子中的分布及氢氧化钠脱毒效果分析

为明确苹果锈果类病毒在八棱海棠果实和种子中的分布特征、种传率以及药剂脱除效果,以带毒母株上的八棱海棠果实和种子为试材,运用RT-PCR技术分析了八棱海棠果实不同部位锈果类病毒的带毒率、实生后代的带毒情况以及氢氧化钠脱除病毒的效果。结果表明,八棱海棠果皮、果肉、种子以及种胚均不同程度携带苹果锈果类病毒,其带毒率分别为96.0%、96.0%、52.0%和4.0%;该病毒可经种子传递给后代,种传率为12.1%;经2%氢氧化钠溶液浸种10、15、20 min,种子的病毒检出率均为0,但后代实生苗的带毒率分别为2.5%、1.3%和0。表明苹果锈果类病毒可侵染种子不同部位并经种子传递给后代,氢氧化钠浸种是脱除该病毒的有效方法。     

Detection of lethal yellowing phytoplasma in embryos from coconut palms infected with Cape St Paul wilt disease in Ghana

This study investigated the potential of seed transmission of Cape St. Paul wilt disease (CSPWD) in coconuts. PCR amplification was used to assess the distribution of phytoplasmas in parts of West African Tall (WAT) palms infected with CSPWD. Employing phytoplasma universal primer pair P1/P7 in standard PCR, or followed with a nested PCR using CSPWD–specific primer pair G813f/AwkaSR, phytoplasma infection was detected in the trunks, peduncles, spikelets, male and female flowers of four infected WAT coconut palms. Through nested PCR, phytoplasma was also detected in four of 19 embryo DNA samples extracted individually from fruits harvested from three of the four infected palms and was confirmed as CSPWD by cloning and sequencing. Subsequently, CSPWD phytoplasma was again detected in five of 33 embryos from nine infected palms, and in one of eight fruits from two symptomless palms. Fruits from infected palms recorded higher percentage germinations in two field nurseries (average of 71·0%) compared to fruits from healthy palms (average of 57·6%), and matured fruits that had dropped from infected palms showed the same levels of germination as those harvested directly from the palms. This indicates that infected fruits retain the ability to germinate whether harvested or dropped. No phytoplasmas were detected in any of the resulting seedlings and plantlets obtained through embryo in-vitro culture. Therefore, although phytoplasma DNA can be detected in embryos, there is as yet no evidence that the pathogen is seed transmitted through to the seedling to cause disease in progeny palms.     

Rice seedbeds as a source of primary infection by Rice yellow mottle virus

The effect of contamination of rice seedlings by Rice yellow mottle virus (RYMV) in seedbeds on the onset and spread of rice yellow mottle in the field was investigated. Rice seedlings were artificially contaminated in seedbeds at different rates (0.1, 0.5, and 2.5%) and pooled in bundles before transplantation, as done by farmers. RYMV was successfully transmitted through contaminated hands and bundling healthy and diseased seedlings together. Hand contamination was responsible for 4.5% infection. Disease incidence in the field after secondary spread reached 32% for 2.5% seedbed contamination rate but remained limited (less than 10%) for all other rates. Eradicating infected plants from seedbeds lessened disease incidence in the field. This technique may be used in conjunction with other prophylactic measures to efficiently control rice yellow mottle disease.     

Transmission of ‘Candidatus Liberibacter solanacearum’ in carrot seeds

A protocol for the specific detection and quantification of ‘Candidatus Liberibacter solanacearum’ in carrot seeds using real‐time PCR was developed. The bacterium was detected in 23 out of 54 carrot seed lots from 2010 to 2014, including seeds collected from diseased mother plants. The average total number of ‘Ca. L. solanacearum’ cells in individual seeds ranged from 4·8 ± 3·3 to 210 ± 6·7 cells per seed from three seed lots, but using propidium monoazide to target live cells, 95% of the cells in one seed lot were found to be dead. Liberibacter‐like cells were observed in the phloem sieve tubes of the seed coat and in the phloem of carrot leaf midrib from seedlings. The bacterium was detected as early as 30 days post‐germination, but more consistently after 90 days, in seedlings grown from PCR positive seed lots in an insect‐proof P2 level containment greenhouse. Between 12% and 42% of the seedlings from positive seed lots tested positive for ‘Ca. L. solanacearum’. After 150 days, symptoms of proliferation were observed in 12% of seedlings of cv. Maestro. ‘Candidatus Liberibacter solanacearum’ haplotype E was identified in the seeds and seedlings of cv. Maestro. No phytoplasmas were detected in seedlings with symptoms using a real‐time assay for universal detection of phytoplasmas. The results show that to prevent the entry and establishment of the bacterium in new areas and its potential spread to other crops, control of ‘Ca. L. solanacearum’ in seed lots is required.     

Apple necrotic mosaic virus,a novel ilarvirus from mosaic-diseased apple trees in Japan and China

The causal agent of apple mosaic disease has been previously thought to be solely caused by apple mosaic virus (ApMV). In this study, we report that a novel ilarvirus is also associated with apple mosaic disease. Next-generation sequencing analysis of an apple tree showing mosaic symptoms revealed that the tree was infected with three apple latent viruses (apple stem pitting virus, apple stem grooving virus, and apple chlorotic leaf spot virus) and a novel ilarvirus (given the name apple necrotic mosaic virus (ApNMV)) that is closely related to Prunus necrotic ringspot virus (PNRSV) and ApMV. The genome of ApNMV consists of RNA1 (3378 nt), RNA2 (2767 nt), and RNA3 (1956 nt). A phylogenetic analysis based on the coat protein amino acid sequences indicated that the novel virus belongs to the same subgroup 3 of the genus Ilarvirus as PNRSV and ApMV. The presence of mosaic leaves, which tend to be unevenly distributed in diseased apple trees, was correlated with the internal distribution of ApNMV. RT-PCR detection of mosaic-diseased apple trees in Japan indicated that ApNMV was detected in apple trees introduced from China, whereas ApMV was detected from cultivated apple trees in domestic orchards. Consistent with these findings, a survey of mosaic-diseased apple trees in major apple-producing provinces in China revealed that the majority of apple trees showing mosaic symptoms in China are infected with ApNMV.     

Fireblight in Békés County (Hungary) in 1996/20021

Fireblight (Erwinia amylovora) appeared in Hungary in 1996. Most damage occurred on apple, pear, quince and medlar, and also on the ornamentals Pyracantha, Sorbus, Cotoneaster and Crataegus. In 1996–2006, an official programme for elimination of infected parts of plants started in Békés county. This mainly concerned trees in towns and villages, since there are few pome‐fruit orchards in the county. Work teams under official direction pruned back or cut down trees. In total, some 13 000 trees were pruned back and nearly 11 000 were cut down. In addition, 21 villages were subjected to special phytosanitary measures. Infection decreased considerably between 1996 and 2002, but over 90% of the inhabited areas in the county remained subject to special measures, because of the very dispersed occurrence of fireblight.     

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.     

Presence of Xylella fastidiosa in Sweet Orange Fruit and Seeds and Its Transmission to Seedlings

ABSTRACT Xylella fastidiosa, a xylem-limited bacterium, causes several economically important diseases in North, Central, and South America. These diseases are transmitted by sharpshooter insects, contaminated budwood, and natural root-grafts. X. fastidiosa extensively colonizes the xylem vessels of susceptible plants. Citrus fruit have a well-developed vascular system, which is continuous with the vascular system of the plant. Citrus seeds develop very prominent vascular bundles, which are attached through ovular and seed bundles to the xylem system of the fruit. Sweet orange (Citrus sinensis) fruit of cvs. Pera, Natal, and Valencia with characteristic symptoms of citrus variegated chlorosis disease were collected for analysis. X. fastidiosa was detected by polymerase chain reaction (PCR) in all main fruit vascular bundles, as well as in the seed and in dissected seed parts. No visual abnormalities were observed in seeds infected with the bacterium. However, the embryos of the infected seeds weighed 25% less than those of healthy seeds, and their germination rate was lower than uninfected seeds. There were about 2,500 cells of X. fastidiosa per infected seed of sweet orange, as quantified using real-time PCR techniques. The identification of X. fastidiosa in the infected seeds was confirmed by cloning and sequencing the specific amplification product, obtained by standard PCR with specific primers. X. fastidiosa was also detected in and recovered from seedlings by isolation in vitro. Our results show that X. fastidiosa can infect and colonize fruit tissues including the seed. We also have shown that X. fastidiosa can be transmitted from seeds to seedlings of sweet orange. To our knowledge, this is the first report of the presence of X. fastidiosa in seeds and its transmission to seedlings.     

<Emphasis Type="Italic">Tobacco mosaic virus</Emphasis> Is Transmissible from Tomato to Tomato by Pollinating Bumblebees

Tobacco mosaic virus (TMV) was detected by ELISA, electron microscopy and/or bioassay from bumblebee (Bumbus terrestris), pollen clumps, nest materials and bee-visited anthers of flowers from greenhouses in which tomatoes had been pollinated by bees and were severely infected with TMV. Experimental bee-mediated transmission of TMV in greenhouse tomatoes demonstrated that the bumblebees transported TMV from plant to plant and that they spread the virus in greenhouses. This is the first report describing TMV transmission by bumblebees. Received 11 August 1999/ Accepted in revised form 30 September 1999     

Contamination of internationally traded wheat by herbicide‐resistant Lolium rigidum

As herbicide‐resistant weeds have spread in the agricultural fields of grain‐exporting countries, their seeds could be introduced into other countries as contaminants in imported grain. The spread of resistance genes through seed and pollen can cause significant economic loss. In order to assess the extent of the problem, we investigated the contamination by herbicide‐resistant annual ryegrass (Lolium rigidum) of wheat imported from Western Australia into Japan. Annual ryegrass seeds were recovered from wheat shipments and seed bioassays were conducted to identify resistance to the herbicides that are commonly used in Australia: diclofop‐methyl, sethoxydim, chlorsulfuron, and glyphosate. Nearly 4500 ryegrass seeds were detected in 20 kg of wheat that was imported in both 2006 and 2007. About 35% and 15% of the seeds were resistant to diclofop‐methyl, 5% and 6% were resistant to sethoxydim, and 56% and 60% were resistant to chlorsulfuron in 2006 and 2007, respectively. None was resistant to glyphosate in either year. As the contamination of crops by herbicide‐resistant weeds is probably a common phenomenon, the monitoring of incoming grain shipments is necessary to stem the further spread of herbicide‐resistant weeds into importing countries.     

<Emphasis Type="Italic">Citrus exocortis viroid</Emphasis> transmission through commercially-distributed seeds of <Emphasis Type="Italic">Impatiens</Emphasis> and <Emphasis Type="Italic">Verbena</Emphasis> plants

Surveys of Impatiens and Verbena species in local nurseries in Fredericton, Canada and Verbena species in New Delhi, India showed widespread infection of Citrus exocortis viroid (CEVd) in vegetatively-propagated and seed-grown plants. To determine viroid seed transmission, samples of eight varieties of Impatiens and 11 varieties of Verbena were obtained from four commercial sources. All 19 samples collected contained viroid infection irrespective of variety. The presence of viroid in non-germinated seed was 21%, while the transmission rate in seedlings was 66% in Impatiens walleriana in 2006. Following 2 years of seed storage, the respective figures were 6% and 26%. Similarly, in Verbena x hybrida the presence of viroid in seed was 13% in 2006 with a seed-transmission rate in seedlings of 28%, while the respective figures after 2 years of storage were 5% and 45%.     

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.     

A comprehensive analysis of the Asiatic citrus canker eradication programme in São Paulo state,Brazil, from 1999 to 2009

Asiatic citrus canker (ACC), caused by Xanthomonas citri subsp. citri, has been controlled by exclusion and eradication measures in São Paulo state (SP), Brazil, since it was first detected in 1957. Several protocols of eradication have been used since then, but the protocol applied from 1999 to 2009 was the most successful and well documented of these. During that time, if the incidence of trees with symptoms in a citrus block was ≤0.5%, the infected trees and the symptomless trees within a 30 m radius were removed. However, if the incidence was >0.5%, the entire block was eliminated. Subsequently, inspections were carried out to monitor the affected blocks during quarantine. Although this protocol did not eliminate the disease from SP, it suppressed the incidence of affected blocks to very low annual levels (<0.20%) for a decade. Of over 5000 foci detected, 65.1% were eliminated by removing the block, either when disease was first detected (47.8%) or at a subsequent detection (17.3%), and 28.4% were eradicated by removing trees within a 30 m radius. The latter method was not an effective stand‐alone measure for ACC eradication and the 0.5% threshold was the key factor in suppression of disease statewide. Currently, a re‐emergence of ACC is occurring in SP. The information presented here will aid the establishment of protocols for the eradication of ACC in other citrus‐growing areas with low incidence of the disease or at risk of pathogen introduction.     

<Emphasis Type="Italic">Cylindrocarpon</Emphasis> species associated with apple tree roots in South Africa and their quantification using real-time PCR

Cylindrocarpon species are known to be a component of the pathogen/pest complex that incites apple replant disease. In South Africa, no information is available on apple associated Cylindrocarpon species and their pathogenicity. Therefore, these aspects were investigated. Among the isolates recovered from apple roots in South Africa, four species (C. destructans, C. liriodendri, C. macrodidymum and C. pauciseptatum) were identified using β-tubulin gene sequencing and phylogenetic analysis. This is the first report of C. liriodendri, C. macrodidymum and C. pauciseptatum on apple trees. Cylindrocarpon macrodidymum was the most prevalent. Isolates within each of the four species were pathogenic towards apple seedlings, but varied in their virulence. With a single exception, all isolates were able to induce lesion development on seedling roots. Only 57% of the isolates, which represented all four species, were able to cause a significant reduction in seedling weight and/or height. The greatest seedling growth reductions were caused by two isolates of C. destructans, and one isolate each of C. liriodendri and C. macrodidymum. A quantitative real-time polymerase chain reaction (qPCR) method was developed for simultaneous detection of all four Cylindrocarpon species. qPCR analyses of Cylindrocarpon from the roots of inoculated seedlings showed that the amount of Cylindrocarpon DNA in roots was not correlated to seedling growth reductions (weight and height) or root rot. The qPCR method is, however, very useful for the rapid identification of apple associated Cylindrocarpon species in roots. The technique may also hold potential for being indicative of Cylindrocarpon disease potential if rhizosphere soil rather than roots are used.