Characterization of Citrus tristeza virus isolates in northern Iran

The biological and molecular properties of four Citrus tristeza virus (CTV) isolates isolated from infected Satsuma trees imported from Japan, and growing in citrus groves in northern Iran (Mahdasht orchards, Mazandaran Province), were investigated. CTV-infected samples were collected from sweet orange trees and grafted onto Alemow (Citrus macrophylla Wester) seedlings. On indicator plants, these isolates produced various symptoms including vein clearing and stem pitting on Mexican lime, Alemow, and Citrus hystrix, and yellowing and stunting on sour orange and grapefruit seedlings. Citrus samples were also surveyed for CTV using serological tests. The coat protein (CP) gene of these isolates was amplified using specific primers, yielding an amplicon of 672 bp for all isolates. Sequence analysis showed 98%–99% sequence homology of Iranian isolates with the Californian CTV severe stem-pitting isolate SY568 and 97%–98% homology with the Japanese seedling yellows isolate NUagA. The Iranian isolates were compared by restriction fragment length polymorphism (RFLP) analysis of the CP amplicon for further classification.     

Further observations of almond stem pitting 1

Two disorders of almond, know as 'stem pitting’and‘graft union necrosis (black line)', were described years ago from Puglia (southern Italy). The etiology of these diseases was uncertain, the causal agent unknown and reliable diagnostic methods were not available. Investigations were therefore carried out to identify Prunus species and/or cultivars as possible indicators for a quick and reliable diagnosis. In 1990, two different almond sources (cv. Filippo Ceo) affected by black line and stem pitting were budded onto seedlings of P. persica, P. cerasifera, P. amygdalus cv. Don Carlo, GF305, self-rooted cuttings of GF677 (P. persica×P. amygdalus) and onto Don Carlo and GF305 seedlings that had been already grafted with almond cvs Genco, Tuono and Filippo Ceo. At least two inoculated plants were inspected each year for the presence of symptoms on the woody cylinder after removal of the cortex. Stem pitting developed on P. persica and P. cerasifera seedlings during the first year after graft inoculation. On the grafted plants that had been inoculated, pitting and grooving of the wood were much more pronounced in cvs Filippo Ceo and Genco, with a higher incidence when GF305 was used as rootstock. Clear symptoms were observed (from the first year in cvs Tuono and Genco), regardless of the rootstock, all along the junction line and often not associated with pitting of the rootstock and scion.     

Molecular characterization of Citrus yellow mosaic badnavirus (CMBV) isolates revealed the presence of two distinct strains infecting citrus in India

Citrus yellow mosaic badnavirus (CMBV) is a non-enveloped, bacilliform DNA virus and the etiologic agent of yellow mosaic disease of citrus in India. The disease was initially reported from the southern parts of India and has now spread to other parts of the country. It is a serious disease of sweet orange (Citrus sinensis) in southern India, where it causes significant yield losses. During a recent survey of citrus groves in the Nagpur region, central India, characteristic mosaic symptoms were observed in mandarin orange (Citrus reticulata) and sweet orange. Virus transmission studies, electron microscopy, PCR amplification and sequencing of cloned PCR products from samples showing mosaic symptoms confirmed the presence of a badnavirus. The CMBV–Nagpur isolate could be transmitted to the Rangpur lime (C. limonia) and acid lime (Citrus aurantifolia) by graft inoculation. Sequence analysis of a segment of ORF-III region and intergenic region (IR) of the viral genome revealed that CMBV–Nagpur isolate formed a distinct clade along with some previously reported isolates that are known to infect acid lime and Rangpur lime. CMBV isolates that infect citrus species other than the acid lime and Rangpur lime formed a second clade. Based on the transmission studies and phylogenetic analyses, it was concluded that at least two strains of CMBV exist in India currently.     

Low glyphosate rates do not affect Citrus limonia (L.) Osbeck seedlings

BACKGROUND: Glyphosate is used to control weeds in citrus orchards, and accidental spraying or wind drift onto the seedlings may cause growth arrest owing to metabolism disturbance. Two experiments were carried out to investigate the effect of non‐lethal rates (0, 180, 360 and 720 g AI ha^?1) of glyphosate on four‐month‐old ‘Cravo’ lime, Citrus limonia (L.) Osbeck, seedlings. Photosynthesis and the concentrations of shikimic acid, total free amino acids and phenolic acids were evaluated. RESULTS: Only transitory effects were observed in the contents of shikimate and total free amino acids. No visual effects were observed. CONCLUSION: The present study showed that glyphosate at non‐lethal rates, which is very usual when accidental spraying or wind drift occurs in citrus orchard, did not cause severe metabolic damage in ‘Cravo’ lime seedlings. Copyright © 2009 Society of Chemical Industry     

Preventive and curative activity of postharvest potassium silicate treatments to control green and blue molds on orange fruit

Preventive and curative antifungal activities of postharvest treatments with potassium silicate (PSi) against green (GM) and blue (BM) molds were evaluated on oranges (cvs. ‘Valencia’ or ‘Lanelate’) artificially inoculated in rind wounds with Penicillium digitatum and P. italicum, respectively. The most effective PSi concentration, the effect of fungal inoculum concentration, and the influence of temporal and spatial factors on antifungal activity were assessed in in vivo primary screenings. After 6 days of incubation at 20 °C, significant preventive (treatment before fungal inoculation) and curative (treatment after inoculation) activities against GM and BM were observed with PSi at 90 mM (GM and BM incidence reductions of 23 and 52 %, and 23 and 40 %, respectively). In preventive tests, the effectiveness of PSi was influenced by inoculum concentration (10³, 10⁴, 10⁵, or 10⁶ spores ml^-1), but not by the distance between treatment and inoculation sites (10, 20 or 30 mm). PSi applied about 2 h before inoculation showed higher preventive activity than applied before 24, 48 or 96 h. In order to determine the best dip treatment conditions, PSi at 90 mM was tested at 20 or 50 °C for 60 or 150 s in small-scale trials with ‘Lanelate’ oranges artificially inoculated before or after the treatment and incubated for 7 days at 20 °C. Dips at 20 °C for 60 s were selected and subsequently applied on inoculated ‘Valencia’ oranges stored at 5 °C and 90 % RH for up to 6 weeks. Curative postharvest dips effectively reduced the incidence and severity of both GM and BM during cold storage, while preventive dips significantly reduced the severity but not the incidence. Overall, postharvest PSi treatments showed potential as a new tool to be part of non-polluting strategies to control penicillium decay of citrus fruit.     

Uncontrolled Citrus psorosis virus infection in Citrus sinensis transgenic plants expressing a viral 24K-derived hairpin that does not trigger RNA silencing

Citrus psorosis virus (CPsV) is the causal agent of psorosis disease of citrus. Pineapple sweet orange plants were transformed with a hairpin construct derived from the viral 24k gene (lines ihp24K). Contrary to expectations, these lines did not trigger efficient RNA silencing, and when infected with CPsV they showed a phenotype of exacerbated symptoms with a persistent and homogeneous infection without the recovery observed in non-transgenic plants. Ihp24K lines did not behave similarly when challenged with Citrus tristeza virus. All these results indicate that hypersusceptibility is likely related to the specific action of 24K-derived hairpin over CPsV multiplication.     

Detection and characterization of <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> stem pitting isolates in Jamaica

An island wide survey for Citrus tristeza virus (CTV) in citrus orchards across Jamaica (13 regions) was conducted over 2 years. Trees (1, 885) showing virus-like symptoms as well as asymptomatic trees were randomly sampled for testing by ELISA and 55 samples from the 6 major citrus growing regions were graft inoculated on indicator plants. Most samples (74%) reacted to polyclonal antibodies against CTV in ELISA, while 20% were positive in tests using monoclonal antibodies specific to severe CTV strains. Samples collected from the 6 major citrus growing regions produced vein clearing and stem pitting symptoms on Mexican lime indicator plants (87%). In addition, stem pitting symptoms were induced on Duncan grapefruit, sweet orange, sour orange or sweet orange grafted on sour orange. Nucleotide sequencing of the coat protein gene sequences isolated from these samples indicated high identities (88 to 95.5%) among the Jamaican isolates and previously reported stem pitting strains from Central and North America and Eurasia (88 to 100%). The results suggest a shared ancestry with isolates from other geographical locations, rather than geographical speciation, and presumably separate CTV introductions into Jamaica.     

A new method for inoculation of fruit with <Emphasis Type="Italic">Guignardia citricarpa</Emphasis>, the causal agent of citrus black spot

Citrus black spot (CBS) is a fungal disease, caused by Guignardia citricarpa, that has a high economic impact on citrus. Although G. citricarpa has been associated with black spot of citrus, an adequate pathogenicity test is still not available. Thus, our objective was to develop and evaluate a simple, safe, and practical pathogenicity test. We used fruits from Pera-Rio and Valencia sweet orange trees from two different orchards, located in the State of São Paulo, Brazil. Inoculation was performed by placing six disks colonized by G. citricarpa, onto the peel of healthy fruits, previously bagged. In the Pera-Rio sweet orange grove, initial symptoms of the false melanose type resulting from the inoculations were observed 55 days after inoculation (dai). In the Valencia grove, initial symptoms also of the false melanose type resulting from the inoculations occurred 73 dai. A total of 92.8% and 86.6% of the Pera Rio and Valencia fruits inoculated, respectively, showed symptoms of CBS. Citrus black spot symptoms were not observed in any of the control fruits.     

Affordable and reliable plant sap-mediated template preparation for the detection of various phytopathogens by PCR assay

Plant pathogens like ‘Ca. Liberibacter’, phytoplasma, viruses and viroids cause diseases to almost all economically important plants. A simple and fast sap-mediated polymerase chain reaction (PCR) method for the detection of these pathogens infecting various plant hosts is described in the present study. Sap from selected plants was drawn aseptically on parafilm, from the mid-rib of young leaves. Depending on the type of host plant, sap was diluted to optimal concentration before PCR analysis. ‘Ca. Liberibacter’, Citrus tristeza virus (CTV) and Citrus exocortis viroid (CEVd) infecting citrus, and ‘Ca. Phytoplasma’ infecting pepper and sandal trees were tested by sap-mediated PCR. The reliability of this procedure was evaluated by comparing the findings with previously described protocols. The sap-mediated nucleic acid template preparation for PCR assay is devoid of laborious nucleic acid extraction and expensive chemicals. Hence the present method is rapid, economical and so can be employed for diagnosis of large number of plant samples.     

A combined indexing and budwood propagation method for citrus

Several budwood-transmitted citrus diseases, including citrus tristeza virus, citrus psorosis, citrus impietratura and a range of citrus viroids, were tested both visually and biochemically on a combined indicator (CInd) plant consisting of an Alemow (Citrus macrophylla) rootstock grafted with Etrog citron (C.medica) and Sour orange (C.aurantium) or Sweet orange (C.sinensis) buds. Indexing on CInd plants is economical for limited testing space; an additional advantage is that, by collecting budwood directly from the CInd plants, the risk of diagnostic failure due to uneven pathogen distribution in the budwood source tree is considerably reduced.     

Evidence for resistance to Citrus tristeza virus in pomelo (Citrus maxima Merr.) grown in Darjeeling and Sikkim hills of India

Darjeeling and Sikkim hills of India are well known for production of mandarin orange (Citrus reticulata). The recent spread of Citrus tristeza virus (CTV) has threatened the citrus cultivation in this region. During a survey in Darjeeling and Sikkim hills, pomelo trees were recorded as CTV free. Since pomelo trees did not show any disease appearance, a study was undertaken to ascertain whether they are resistant to CTV infection or resistant to aphid feeding or both. Toxoptera citricida, the most efficient aphid vector and which is abundantly present in this region, did not feed on pomelo when other Citrus species such as mandarin, kagzi lime and rough lemon were available. Additionally, CTV isolates of Darjeeling and Sikkim hills were not transmissible to pomelo either by T. citricida or grafting. We report for the first time that pomelo is resistant to isolates of CTV present in this region.     

Influence of temperature on infection and establishment of ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’ in citrus plants

The objectives of this work were (i) to determine the influence of temperature on infection of citrus by ‘Candidatus Liberibacter asiaticus’ and ‘Candidatus Liberibacter americanus’, the two bacterial species associated with citrus huanglongbing (HLB) in Brazil, and (ii) to determine the influence of temperature on citrus colonization by ‘Ca. L. asiaticus’, which has taken over from ‘Ca. L. americanus’ as the predominant species in Brazil since 2008. Two experiments were carried out with graft‐inoculated Valencia oranges on Rangpur lime rootstocks. Immediately after inoculation the plants were maintained for 423 days in growth chambers under the following night/day temperature conditions: 17/22, 22/27 or 27/32°C, with a dark/light photoperiod of 8/16 h. Infection and colonization of plants were determined using quantitative PCR (qPCR). ‘Candidatus Liberibacter americanus’ did not infect the plants maintained at 27/32°C; however, infection by ‘Ca. L. asiaticus’ occurred at all studied temperatures. Two months after inoculation, ‘Ca. L. asiaticus’ was distributed throughout the inoculated plants, with mean C_t values in the range of 30–31 for leaves and 25–28 for roots. Over time, ‘Ca. L. asiaticus’ reached the highest titres in mature leaves (mean C_t value = 26·7) of citrus plants maintained at 22/27°C. ‘Candidatus Liberibacter asiaticus’ colonization of citrus plants was negatively affected by the daily temperature regime of 27/32°C (mean C_t value in mature leaves = 33·6).     

Photosynthetic and antioxidant responses of Mexican lime (Citrus aurantifolia) plants to Citrus tristeza virus infection

The effect of Citrus tristeza virus (CTV) infection on photosynthetic activity and antioxidant metabolism was analysed in plants of the highly susceptible citrus genotype Mexican lime (Citrus aurantifolia). Two virus isolates differing in their virulence (the severe T318 and the mild T385) were used in the experiments. CTV infection caused a reduction in photosynthetic capacity in infected plants. This limitation was mainly due to a reduction in the carboxylative efficiency whereas the limitation of CO₂ diffusion through the stoma had lower impact. The virus did not damage the antennae and did not reduce the efficiency of light harvesting complexes. Oxidative damage occurred in infected plants, as evidenced by the increase in malondialdehyde levels. Indeed, CTV infection caused an increase in ascorbate peroxidase activity in new shoots developed in infected plants during the 2 years of the experiment. Data suggest that the H₂O₂ removal machinery was not damaged as a result of stress but the defence mechanism was overwhelmed with time due to the continuing pressure of biotic stress.     

Expression of Endochitinase from Trichoderma harzianum in Transgenic Apple Increases Resistance to Apple Scab and Reduces Vigor

ABSTRACT The goal of this research was to improve scab resistance of apple by transformation with genes encoding chitinolytic enzymes from the bio-control organism Trichoderma harzianum. The endochitinase gene, as cDNA and genomic clones, was transferred into apple cv. Marshall McIntosh by Agrobacterium-transformation. A total of 15 lines were identified as transgenic by NPTII enzyme-linked immunosorbent assay and polymerase chain reaction and confirmed by Southern analysis. Substantial differences in endochitinase activity were detected among different lines by enzymatic assay and western analysis. Eight lines propagated as grafted and own-rooted plants were inoculated with Venturia inaequalis. Six of these transgenic lines expressing endochitinase were more resistant than nontransformed cv. Marshall McIntosh. Disease severity compared with cv. Marshall McIntosh was reduced by 0 to 99.7% (number of lesions), 0 to 90% (percentage of leaf area infected), and 1 to 56% (conidia recovered) in the transgenic lines tested. Endochitinase also had negative effects on the growth of both inoculated and uninoculated plants. There was a significant negative correlation between the level of endochitinase production and both the amount of disease and plant growth.     

Survey of <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> (CTV) diversity in pigmented <Emphasis Type="Italic">Citrus x paradisi</Emphasis> (Macfad.) (Grapefruit) trees in north-western Argentina

Citrus tristeza virus (CTV) is the most severe viral pathogen of citrus and elicits a wide range of devastating disease symptoms. Grapefruit cultivars (Citrus x paradisi) are the most sensitive among citrus to the effects of CTV infections. Grapefruit is an important crop within the north-western Argentine citrus industry; however, production has been affected by CTV stem-pitting. In general, CTV diversity within South America is poorly studied, with data on grapefruit CTV populations being particularly limited. In this study, 50 samples were collected from Star Ruby, Henninger’s Ruby and Ruben Pink cultivars, within the provinces of Tucumán, Salta and Jujuy in north-western Argentina. The CTV p33 gene was PCR amplified and the resulting amplicons sequenced with Sanger sequencing. A subset of these amplicons was sequenced with Illumina MiSeq sequencing. AT-1-like sequences were dominant within the majority of populations, as determined by Sanger sequencing, followed by sequences clustering within the unresolved Kpg3/SP/T3 and RB clades. Sequencing by Illumina MiSeq confirmed this, as well as detecting minor sequence types within the HA 16–5, VT, B165 and A18 clades.     

酵母pac1基因介导对葡萄B病毒的抗性

为明确广谱性抗病毒基因—酵母pac1基因对葡萄B病毒(Grapevine virus B,GVB)的抗性效果,通过农杆菌介导的遗传转化,将pac1基因导入西方烟37B,对转基因植株进行PCR鉴定及Southern blot分析,通过病毒摩擦接种观察症状以及实时荧光定量RT-PCR检测植株体内病毒含量,并对转基因植株抗病性进行初步鉴定。结果表明,目的基因pac1成功导入并整合至西方烟37B基因组,共获得10个转基因株系。不同株系的T1代烟草中阳性植株比例为16.7%~72.7%,表明目的基因可成功遗传到子代。接种病毒后转基因植株普遍延迟发病,但后期症状与非转基因对照相似,其中仅1个转基因株系B6具有不表现典型症状等抗性反应。接种植株病毒含量检测中,所有转基因植株均检测到病毒存在,但表现为抗病的B6株系中病毒含量显著低于非转基因对照,表明该转基因植株虽不能完全抵抗GVB侵染,但对GVB具有耐病性。     

Protection of Citrus Rootstocks Against Phytophthora spp. with a Hypovirulent Isolate of Phytophthora nicotianae

ABSTRACT Phytophthora root rot of citrus in Florida is caused by Phytophthora nicotianae and P. palmivora. A naturally occurring isolate of P. nicotianae (Pn117) was characterized as hypovirulent on citrus roots. Pn117 infected and colonized fibrous roots, but caused significantly less disease than the virulent isolates P. nicotianae Pn198 and P. palmivora Pp99. Coincident inoculation of rootstock seedlings of Cleopatra mandarin (Citrus reticulata) or Swingle citrumelo (C. paradisi x Poncirus trifoliata) with the hypovirulent Pn117 and the virulent isolates Pn198 and Pp99 did not reduce the severity of disease caused by the virulent Phytophthora spp. When either rootstock was inoculated with the hypovirulent Pn117 for 3 days prior to inoculation with virulent isolates, preinoculated seedlings had significantly less disease and greater root weight compared with seedlings inoculated with the virulent isolates alone. Recovery of the different colony types of Phytophthora spp. from roots of sweet orange (C. sinensis) or Swingle citrumelo was evaluated on semiselective medium after sequential inoculations with the hypovirulent Pn117 and virulent Pp99. Pn117 was isolated from roots at the same level as the Pp99 at 3 days post inoculation. Preinoculation of Pn117 for 3 days followed by inoculation with Pp99 resulted in greater recovery of the hypovirulent isolate and lower recovery of the virulent compared with coincident inoculation.     

Effects of resistance of Eremocitrus glauca and Microcitrus australis to viroid infection: replication,accumulation and long‐distance movement of six citrus viroids

In studies to identify genotypes resistant to infection with citrus viroids, Eremocitrus glauca and Microcitrus australis were selected because their evolution in their habitat in Australia and New Guinea may have led to the selection of unusual traits. The movement and accumulation of Citrus exocortis viroid (CEVd), Hop stunt viroid, Citrus bent leaf viroid, Citrus dwarfing viroid, Citrus bark cracking viroid and Citrus viroid V (CVd‐V) in self‐rooted as well as in graft‐ propagated E. glauca and M. australis plants was assessed by northern hybridization, RT‐PCR and by topworking to the sensitive selection 861‐S1 of Etrog citron. In both plant species the inoculated viroids were undetectable unless these plants were grafted to a susceptible Citrus partner, the rough lemon rootstock and/or the topworked Etrog citron, which acted as viroid sources. The results obtained indicate that M. australis and in particular E. glauca are poor viroid hosts in which viroid replication/accumulation does not occur or is extremely inefficient. However, viroid downward and upward movement to grafted Citrus partners in which viroid replication and accumulation occurs efficiently was not impaired. Eremocitrus glauca and M. australis showed differences regarding their properties as viroid hosts, but for both species CEVd seemed to have the lowest affinity among the viroid species tested and CVd‐V the highest. Even though E. glauca and M. australis do not appear to be truly resistant to viroid infection, they are interesting genotypes for further characterization of the mechanisms involved in viroid infection.