Goat horns: Platforms for viroid transmission to fruit trees?

Mechanical inoculations with contaminating tools and propagation of infected budwood were considered the main causes for the omnipresence of multiple viroid species among citrus and other Middle Eastern and Mediterranean fruit trees and grapevines. However, neither means could explain viroid infections of wild trees — scattered on terrains inaccessible to humans — nor the finding of similar viroids among graft-incompatible plants. Northern hybridization of RNA extracts made of scrapings from the surfaces of goat (Capra hircus) horns that were rubbed against etrog (Citrus medica) stems infected with a citrus viroids complex, revealed accumulation of considerable amounts ofCitrus exocortis viroids (CEVd) andHop stunt viroids (HSVd). Experimental transmission of both CEVd and HSVd was obtained by rubbing healthy citrus plants with goat horns that had been rubbed 24 h earlier on infected etrog stems. These results implicate goats as possible vectors of viroids. Transmissionvia goats could have facilitated the long-range spread of viroids among cultivated and wild plants andvice versa and also among graft-incompatible plants.     

Molecular detection and quantification of Colletotrichum abscissum in sweet orange propagative material

Postbloom fruit drop disease (PFD) has caused serious impacts on citrus production in the Americas, occurring sporadically and suddenly during rain in the flowering period. In São Paulo State, Brazil, Colletotrichum abscissum is the causal agent responsible for over 80% of the disease incidence. Pathogen dispersal over long distances and the origin of primary inoculum are still unclear for PFD epidemics. We tested the hypothesis that citrus propagation material can harbour C. abscissum DNA by quantifying it in leaves of budwood increase block trees (BIB) and young citrus plants (YP) using multiplex quantitative PCR (qPCR). C. abscissum DNA was detected in all citrus nurseries, regardless of the type of propagative material, the sweet orange variety, or the nursery location. Overall, 73.4% of all samples from citrus nurseries have DNA from the pathogen, with a detection limit of 10 conidia. The average of 155 conidia found in YP was higher than the conidia observed on leaf samples from BIB (p = 0.03), although leaf samples from cultivars Valencia and Pera did not differ significantly, with means around 127 and 118 conidia, respectively (p = 0.75). This is the first molecular detection of C. abscissum in citrus propagative material. The multiplex qPCR assay may be used as a protocol for an accurate diagnosis of C. abscissum in citrus propagative material, may assist a better understanding of the pathogen dispersal over long distances, and may be used for further studies involving the quantification of C. abscissum in citrus orchards.     

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.     

Identification and characterization of known and novel viroid variants in the Greek national citrus germplasm collection: threats to the industry

Seventy four percent of the budwood tree sources samples from the Greek national citrus germplasm foundation collection were positive for one or more viroids. Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), the two potentially damaging viroids for the Greek citriculture, especially after transitioning to Citrus tristeza virus resistant/tolerant rootstocks and scions, were detected along with Citrus bark cracking viroid, Citrus bent leaf viroid (CBLVd), and Citrus dwarfing viroid (CDVd). All samples tested negative for Citrus viroid V (CVd-V), CVd-VI and CVd-I-LSS (CBLVd variant). An HSVd isolate related to the non-cachexia variant contained two critical cachexia-related nucleotide changes, while two more isolates were unique among the previously reported HSVds. Unusual CDVd isolates with altered RNA secondary structure were identified in trees additionally co-infected with CEVd and HSVd. Budwood sources that had previously undergone therapy tested negative for all targeted viroids, suggesting that budwood sources in Greece can be protected against graft-transmissible pathogens, even under severe inoculum pressure. Therapied and tested citrus propagative material requires a comprehensive program not available currently in Greece, involving regulators, scientists, and the private sector, for the establishment and successful operation of a national citrus germplasm collection.     

Enhanced resistance to citrus canker in transgenic sweet orange expressing the sarcotoxin IA gene

Citrus canker, caused by the bacterial pathogen Xanthomonas citri subp. Citri (Xcc), is a serious disease reported in most citrus-producing areas around the world. Although different levels of field resistance to citrus canker have been reported in sweet oranges, they are usually not sufficient to provide adequate control of the disease. Ectopic over-expression of antibacterial genes is one of the potential strategies to increase plant resistance to bacterial diseases. Previous in vitro results showed that sarcotoxin IA, an antimicrobial peptide isolated from the flesh fly (Sarcophaga peregrina), can be efficient to control different plant pathogenic bacteria, including Xcc. Transgenic “Pera” sweet orange (Citrus sinensis [L.] Osbeck) plants constitutively expressing the sarcotoxin IA peptide fused to the PR1a signal peptide from Nicotiana tabacum for secretion in the intercellular space were obtained by Agrobacterium-mediated transformation using thin sections of mature explants. Citrus canker resistance evaluation in leaves of transgenic and non-transgenic plants was performed through inoculations with Xcc by infiltration and spraying. The Xcc population was up to 2 log unit lower in leaves of the transgenic plants compared to those of non-transgenic controls. Incidence of canker lesions was significantly higher in non-transformed controls (>10 lesions/cm²) than in the transgenic plants (<5 lesions/cm²) after injection infiltration or spraying with Xcc inoculum. Accumulation of sarcotoxin IA peptide in sweet orange tissue did not cause any deleterious effects on the growth and development of the transgenic plants, indicating this approach is suitable to provide resistance to citrus canker.     

Effect of temperature on RNA silencing of a negative‐stranded RNA plant virus: Citrus psorosis virus

Citrus psorosis virus (CPsV), genus Ophiovirus, causes a bark scaling disease of citrus. CPsV virions are kinked filaments with three negative‐stranded RNA molecules (vRNA) and a 48 kDa coat protein. The effect of temperature on symptom expression, virus accumulation and RNA silencing was examined in sweet orange seedlings (Citrus sinensis) graft‐inoculated with three different CPsV isolates and grown in a glasshouse at 26/18°C or 32/26°C (day/night). Most plants kept in the cooler glasshouse showed a shock reaction in the first flush with shoot necrosis, and then moderate to intense chlorotic flecking and spotting in young leaves, whereas plants incubated at 32/26°C did not exhibit shoot necrosis, and young leaf symptoms were milder. Virus titre estimated by ELISA and by northern and dot blot hybridization paralleled symptom intensity, with significantly higher virus accumulation in plants incubated at 26/18°C. The amount of CPsV‐derived small RNAs (CPsV‐sRNAs) slightly increased at 32/26°C, with the ratio of CPsV‐sRNA/vRNA being higher at 32/26°C than at 26/18°C. These results suggest that (i) CPsV infection induces RNA silencing in citrus plants, (ii) symptom intensity is associated with virus accumulation, and (iii) temperature increase enhances the RNA silencing response of citrus plants and decreases virus accumulation.     

Identification of the Chinese Box Orange (Severinia Buxifolia) as an Alternative Host of the Bacterium Causing Citrus Huanglongbing

The Chinese box orange (Severinia buxifolia) was shown by graft-inoculation and psyllid-transmission tests to be an alternative host of the bacterium causing citrus Huanglongbing (HLB). A PCR-based assay for detection of the HLB bacterium (HLBB) was used to monitor HLBB. In graft-inoculation tests, the Chinese box orange (CBO) grafted with HLBB-infected scions of Luchen sweet orange (LSO) were positive for HLBB, 2–3 months after grafting. The back-grafting test demonstrated that HLBB-infected CBO scions could transmit HLBB back to LSO hosts via grafting. In psyllid-transmission tests, psyllids (insect vectors) transmitted HLBB to CBO plants, in which HLBB could be detected 3–4 months after inoculation. Acquisition-access tests of psyllids revealed that HLBB-free psyllids can acquire HLBB from diseased CBO hosts and can transmit HLBB back to the LSO plants. A field survey verified the presence of HLBB-infected CBO plants in the vicinity of citrus orchards. In this paper, CBO is shown to be a susceptible host plant in which HLBB can exist and replicate. It is also a donor plant from which HLBB can be transmitted to citrus hosts by grafting or by psyllid vectors.     

Effect of different host plants on the development time and parasitization rate of eretmocerus dehachi rose and rosen (hymenoptera: aphelinidae) withparabemisia myricae (Kuwana) (homoptera: aleyrodidae) as insect host

The effect of six citrus and five non-citrus host plants on the developmental time ofEretmocerus dehachi Rose and Rosen (Hymenoptera: Aphelinidae), the parasitoid ofParabemisia myricae (Kuwana) (Homoptera: Aleyrodidae), was studied under laboratory conditions. On citrus host plants, the mean developmental time from egg to adult was shortest on lemon with 14.6 days and longest on trifoliate orange with 26.3 days. On non-citrus host plants the developmental time ranged between 16.3 days on vine and 23.0 days on pomegranate. Among the various host plants tested,E. dehachi preferredP. myricae on sweet orange. A high mortality ofP. myricae due to host feeding and parasitization was observed on sweet orange and grapefruit among citrus host plants and on rose among non-citrus host plants.     

Effect of different host plants on the development time and parasitization rate of eretmocerus dehachi rose and rosen (hymenoptera: aphelinidae) withparabemisia myricae (Kuwana) (homoptera: aleyrodidae) as insect host

The effect of six citrus and five non-citrus host plants on the developmental time ofEretmocerus dehachi Rose and Rosen (Hymenoptera: Aphelinidae), the parasitoid ofParabemisia myricae (Kuwana) (Homoptera: Aleyrodidae), was studied under laboratory conditions. On citrus host plants, the mean developmental time from egg to adult was shortest on lemon with 14.6 days and longest on trifoliate orange with 26.3 days. On non-citrus host plants the developmental time ranged between 16.3 days on vine and 23.0 days on pomegranate. Among the various host plants tested,E. dehachi preferredP. myricae on sweet orange. A high mortality ofP. myricae due to host feeding and parasitization was observed on sweet orange and grapefruit among citrus host plants and on rose among non-citrus host plants.     

Reaction of transgenic Citrus sinensis plants to Citrus tristeza virus infection by Toxoptera citricida

Transgenic Citrus sinensis ‘Hamlin’ and ‘Valencia’ plants containing Citrus tristeza virus (CTV)-derived sequences were propagated and inoculated with CTV. For propagation, selected buds from transgenic and non-transgenic control plants were grafted onto C. aurantium and C. limonia rootstock plants. CTV inoculation was performed via viruliferous aphids (Toxoptera citricida), and viral detection post-inoculation was performed through DASI-ELISA or RT-qPCR. After four inoculations, none of the transgenic lines tested showed complete resistance. However, viral multiplication was undetectable in some of the propagated clones. These resistant clones mainly came from transgenic ‘Valencia’ sweet orange plants grafted onto C. limonia rootstock containing the pCTV-CS gene construct. Although the tested viral inoculation method represents natural field infection conditions, the results did not differ significantly from those previously reported when the same transgenic lines were bud-graft inoculated. This finding indicates that the difficulties in producing CTV-resistant transgenic citrus lines may be unrelated to the inoculation method. Transgene expression level was quantified by RT-qPCR analysis and it was not possible to relate transgene mRNA level with resistance to the pathogen.     

Honeybees can spread Colletotrichum acutatum and C. gloeosporioides among citrus plants

Postbloom fruit drop (PFD) is an important citrus disease that causes up to 100% yield losses during years in which conditions are favourable for the occurrence of epidemics. The conidia of Colletotrichum acutatum and C. gloeosporioides, causal agents of PFD, are predominantly dispersed by rain splash. At the beginning of epidemics, the distribution of diseased plants is random and the disease progress rate is very high, which is unusual for pathogens spread by rain splash. As the pathogen produces abundant conidia on diseased petals, pollinating insects may contribute to disease dispersal. This study investigated honeybees (Apis mellifera) as dispersal agents of C. acutatum and C. gloeosporioides among citrus plants. Two experiments were carried out in a screenhouse in which citrus plants were protected (or not) in insect‐proof cages. The source of inoculum was placed on one side of the screenhouse, and a honeybee hive was placed on the opposite side. All uncaged plants showed symptoms of the disease, and none of the caged plants exhibited PFD symptoms. The monomolecular model showed a good fit to disease progress in both experiments. Conidium‐like structures of Colletotrichum spp. were identified attached to the bodies of the honeybees by scanning electron microscopy. These results have revealed that honeybees disperse Colletotrichum among citrus plants.     

Phyllocnistis citrella and its parasitoids in three citrus species in Greece

The relative abundance as well as the percentage of parasitism of Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae) and its parasitoids were studied in a citrus orchard with orange, mandarin and lemon trees in Greece. Infestation of P. citrella on each citrus host, as expressed by the number of mines per leaf, was significantly higher on lemon than on mandarin. The P. citrella parasitoid complex included the native species Neochrysocharis formosa and Pnigalio pectinicornis, as well as the introduced Citrostichus phyllocnistoides. The most abundant of those was N. formosa in all of the citrus species. The average percentage of parasitism was 13.1%, 13.8% and 11.7% on orange, mandarin and lemon, respectively. No significant differences in parasitism rate by each of the three parasitoid species separately were recorded among the three citrus species.     

Effect of vesicular arbuscular mycorrhiza on citrus growth and mineral composition

The vesicular arbuscular fungusGlomus mosseae (Nicol. & Gerd.) Gerd. & Trappe was retrieved from citrus trees growing in loess soil in the Negev region of Israel. Inoculation of citrus seedlings with the mycorrhizal fungus greatly increased the growth of plants in soil low in phosphorus. Rough lemon responded more than Sour orange. Vesicular arbuscular mycorrhiza caused higher concentrations of P and Cu and lower concentrations of N, K and Ca in leaves of inoculated plants.     

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.     

Presence of the invasive whitefly Aleurocanthus spiniferus (Hemiptera: Aleyrodidae) in Greece

Aleurocanthus spiniferus (Quaintance) (Hemiptera: Aleyrodidae), commonly known as the citrus (or orange) spiny whitefly, is an important pest of various economic crops such as citrus and tea and causes severe economic losses. It is reported for the first time in Greece, in the island of Corfu (North‐West Greece) on sweet orange trees (Citrus sinensis). Morphological and molecular data has been used for species identification. Sequences of the mitochondrial cytochrome oxidase I (COI) gene from Greek populations have been compared with sequences of the species from Italy and Montenegro, as well as with COI sequences retrieved from GenBank, to examine the genetic diversity of the species. Based on the preliminary results, it appears that the population from Corfu probably arose through several independent introductions.     

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.     

Laboratory transmission of the citrus stubborn disease agent by a leafhopper from theCirculifer tenellus complex from the Jordan Valley

Citrus stubborn disease (=little leaf disease) is known to affect the size of citrus trees, and to reduce the quality and quantity of the fruit. The disease agent,Spiroplasma citri, was isolated and culturedin vitro from Oroblanco, orange and grapefruit orchards throughout the year from different regions in Israel. The agent was transmitted by a leaf-hopper from theCirculifer tenellus complex collected onAtriplex halimus plants in the southern Jordan Valley. The latent period of the agent in this vector was at least 10 days following a 3-day acquisition feeding onMatthiola incana plants. It was similar to that found for a primary isolation in culture medium (LP₅₀=21 days). The limitations of visual inspection for recording disease incidence in citrus groves were determined and this method was compared with other methods for detection of the disease agent (immunoassay and cultivation in a culture medium). Plants from various botanical families were tested for their ability to serve as hosts for the Israeli biotype of the beet leaf-hopper and for the stubborn disease agentS. citri. The possible role of IsraeliC. tenellus in the disease epidemiology is discussed.     

Liberibacters associated with orange jasmine in Brazil: incidence in urban areas and relatedness to citrus liberibacters

Two surveys (2005/2006 and 2009) were conducted in the state of São Paulo, Brazil, to investigate the incidence of ‘Candidatus Liberibacter asiaticus’ and ‘Ca. L. americanus’, two liberibacters associated with citrus huanglongbing (HLB) disease and both transmitted by Diaphorina citri, in orange jasmine (Murraya exotica), a widespread ornamental tree in cities and villages. The graft‐transmissibility of the two species, and their DNA relatedness to citrus‐associated liberibacters, were also investigated. Quantitative PCR was applied to PCR‐positive orange jasmine and HLB‐positive citrus growing in backyards and orchards to assess their inoculum source potentials. Liberibacters were detected in 91 of 786 sampled orange jasmine plants in 10 of 76 sampled locations. PCR‐positive trees exhibited yellow shoots and/or dieback symptoms indistinguishable from those on PCR‐negative trees. ‘Candidatus Liberibacter americanus’ was more common in 2005/2006 (96·6%) and ‘Ca. L. asiaticus’ in 2009 (84·8%). rplJ nucleotide sequences were identical within all populations of either species. Graft transmission succeeded only in homologous host combinations, including ‘Ca. L. americanus’ (2/10) from/to orange jasmine and ‘Ca. L. americanus’ (5/18) and ‘Ca. L. asiaticus’ (5/9) from/to citrus. Symptoms were mild and developed less rapidly in orange jasmine than in citrus, probably as a result of lower liberibacter multiplication rates. Respective titres of ‘Ca. L. americanus’ and ‘Ca. L. asiaticus’ in orange jasmine averaged 4·3 and 3·0 log cells g^?1 tissue, compared with 5·5 and 7·3 in citrus. The results indicate that orange jasmine does not favour liberibacter multiplication as much as citrus. However, its importance in HLB epidemics should not be underestimated as it is a preferred host of D. citri and is not under any strict tree‐eradication programme or measures for insect control.     

<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.     

柑桔黄龙病毒研究初报

試驗用萊檬(Citrus aurantifolia)及麻疯柑(C.hystrix)作指示植物,用两广黃龙病区病株接穗嫁接接种,表現脉明、叶脉木栓化、茎部凹斑、及矮化、小叶等典型退化病毒(tristeza virus)所致症状,可知我国柑桔黄龙病与国外柑桔退化病(tristeza)系同一种病毒(或病毒群)所致。无病区的四川江津、湖南溆浦和北京的健康甜橙、桔类、及北京檸檬也带有黄龙病毒,可見黄龙病毒的分布远较病区为广。各种柑桔上所带黄龙病毒株系的強弱不同;在碰柑上的为强毒系,甜橙上的为强毒系或较弱毒系,桔类及北京檸檬上的各别为較弱毒系及弱毒系。