Citrus yellow mosaic badnavirus infecting <Emphasis Type="Italic">Citrus</Emphasis> sp.: a threat to the citrus industry and a quarantine issue

Among the virus diseases affecting the citrus industry in southern parts of India, the most devastation is caused by citrus tristeza virus (CTV) followed by citrus yellow mosaic badnavirus (CMBV). Research analysis reveals that the longevity of the trees, quality of fruit appearance, juice and ascorbic acid content are lowered in CMBV-infected citrus. CMBV is chiefly propagated through infected budwood, which is used in graft propagation of citrus species. Symptoms of the mosaic disease caused by the virus are always confused with those of mineral deficiencies and is the main reason for CMBV spread through graft propagation in nurseries. CMBV could also co-exist as a mixed infection with CTV. This mixed infection of CMBV and CTV causes even more devastation than CTV infection does alone. At present, this virus is limited to the southern parts of India. If CMBV is not properly diagnosed at the quarantine level, there is a high risk of virus movement to other citrus-growing countries of the world, especially for the border countries of India.     

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。     

The influence of citrus rootstocks on the relationship between the mite Brevipalpus phoenicis and citrus leprosis disease

BACKGROUND: Leprosis is one of the most serious citrus plant diseases. Leprosis‐affected plants, especially sweet orange [Citrus sinensis (L.) Osbeck], which is the most widely cultivated citrus fruit worldwide, show reduced photosynthetic capacity and severe defoliation. The aim was to evaluate the relationship between the Brevipalpus phoenicis (Geijskes) vector mite and citrus leprosis disease in Pera sweet orange plants grafted on different rootstocks. Data were analysed using numerical classification and conventional statistical analysis (ANOVA). RESULTS: Both viruliferous and non‐viruliferous B. phoenicis populations increased in number on plants maintained at low soil water content. Among the evaluated rootstocks, Sunki mandarin proved least favourable for mite population increase. Furthermore, the viruliferous mite population increased more rapidly than the non‐viruliferous mite population. CONCLUSION: The Cleopatra rootstock showed low variability in leaf nitrogen content, low mite number and low leprosis severity, thereby producing the most favourable results. Under appropriate moisture conditions, the Sunki rootstock produced the best results, showing the least severe leprosis. Rangpur lime rootstock grown in soil with varying water content showed the greatest variation in foliar nitrogen content, mite number and severity of leprosis and is considered to be the most difficult rootstock to manage. Copyright © 2012 Society of Chemical Industry     

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.     

Separation and interference of strains from a citrus tristeza virus isolate evidenced by biological activity and double-stranded RNA (dsRNA) analysis

Separation of strains of citrus tristeza virus (CTV), differentiated by their double-stranded RNA (dsRNA) profiles, was obtained by graft-inoculating citron plants from a Mexican lime that had been recently aphid- or graft-inoculated with a mild CTV isolate (T-385). Up to 24 sub-isolates with differing dsRNA profiles were obtained from the aphid-inoculated lime. Some of these sub-isolates induced stronger symptoms in several citrus species than the original T-385 isolate. One sub-isolate, T-385-33, was mild in Mexican lime, but induced stem pitting on sweet orange. Inoculation of this isolate on Mexican lime, sour orange and Eureka lemon induced mild or no symptoms when inoculum was taken from citron, but very severe symptoms when the inoculum was from sweet orange. Mexican lime and sweet orange plants co-inoculated with T-385-33 from sweet orange in combination with the other 23 sub-isolates showed mild symptoms. The results obtained suggest that there is natural cross-protection among sub-isolates in the original T-385 isolate.     

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.     

Sequence and evolutionary analysis of ribosomal DNA from Huanglongbing (HLB) isolates of Western India

Citrus greening (Huanglongbing, HLB) is a widespread and economically important citrus disease all over the world. The disease is caused by a phloem-limited fastidious gram negative bacterium, “Candidatus Liberibacter spp.” which belongs to the alpha-proteobacteria group classified on the basis of its 16SrDNA sequence. Although the pathogen has been classified under three distinct groups, viz. Asian, African and American isolates, nothing is known about the status and the molecular variabilities among the Indian HLB isolates collected from different citrus cultivars grown in India. Five different HLB isolates showing variable symptoms based on their severity of infection on different citrus, viz. Mosambi, Rangpur lime, Cleopatra mandarin, acid lime and rough lemon, were studied by PCR amplification, sequence and evolutionary analysis of their 16S and 16S/23S rDNA intergenic regions. Analysis of the 16S/23S rDNA intergenic region separated all five Indian isolates from existing African isolates but failed to differentiate among Asian, American and Indian isolates. However, further analysis of complete 16S rDNA clearly indicated that Indian isolates fall within the Asian HLB group. Overall, our results suggest that all the five Indian HLB isolates taken for the current analysis belong to the Candidatus Liberibacter asiaticus strain, which showed distinct sequence variabilities and produced noticeable symptoms on the citrus trees. These results provide a robust framework for understanding how differences in pathogenicity among various HLB isolates may be related to evolutionary history.     

Biological characterization of citrus tristeza virus isolates by in vitro tissue cultures

Stem segments from Mexican lime, sweet orange, grapefruit, Citrus excelsa and alemow, infected with five citrus tristeza virus (CTV) isolates, were cultured in vitro . Regeneration of roots and shoots were modified as a result of infection. The effect of CTV on the morphogenesis of stem segments cultured in vitro depended on the CTV isolate and the plant host, and showed a correlation with the in vivo effects observed in biological indexing. Evaluation of the morphogenic response of stem segments of Mexican lime and grapefruit can be used as an additional tool for the biological characterization of CTV isolates. The symptoms on sweet orange plants obtained from regenerated shoots indicated that CTV is unevenly distributed in the host plant cells and that the regeneration process may be utilized as a tool to separate strains from complex field isolates.     

Investigation of biological properties, double-stranded RNA patterns and antigen concentration in citrus species infected with citrus tristeza virus

Biological properties and dsRNA patterns of one Cyprus and three Turkish isolates of citrus tristeza virus (CTV) were investigated. In addition, CTV antigen concentration and effect of tissue sampling time from naturally infected Shamouti sweet orange trees grown in the field of Icel Province, Turkey, were also determined. The Cyprus isolate showed vein clearing symptoms on grapefruit, ‘Madam Vinous’ and Mexican lime and stem pitting symptoms on Mexican lime. The three Turkish isolates showed only vein clearing symptoms on Mexican lime. All four isolates showed a full-length major double-stranded RNA (dsRNA) band of 13.3 × 10⁶ Da mol. wt in extracts from infected Madam Vinous sweet orange trees, and major or minor dsRNA bands with 2.0. 0.8 and 0.5 × 10⁶ mol.wt. All seven different citrus varieties inoculated with the Igdir (D) strain contained full-length dsRNA. The additional two dsRNA of 0.8 and 0.5 × 10⁶ mol.wt were also detected as clearly as full-length dsRNA in these hosts, but were weaker inCitrus exelsa and ‘Interdonat’ lemon. Madam Vinous, rough lemon and Mexican lime were the best hosts for dsRNA analysis. ELISA values were highest in April (OD_405nm =0.476), decreased steadily until August, and then increased gradually through December. ELISA values were lowest in July and August (OD_405nm =0.157 and 0.141, respectively). dsRNA recovery from a field tree infected with isolate Igdir D was good in March, April and May and poor in January and February. No dsRNA band was detected in August or September. http://www.phytoparasitica.org posting July 9, 2002.     

Postbloom fruit drop of citrus and key lime anthracnose are caused by distinct phylogenetic lineages of Colletotrichum acutatum

Colletotrichum acutatum causes two diseases of citrus, postbloom fruit drop (PFD) and Key lime anthracnose (KLA). PFD is a disease restricted to flowers of sweet orange and most other citrus, and symptoms include petal necrosis, abscission of developing fruit, and the formation of persistent calyces. KLA is a disease of foliage, flowers, and fruits of Key lime only, and symptoms include necrotic lesions on leaves, fruits, twigs, flowers, and blight of entire shoots. The internal transcribed spacers 1 and 2 and the gene encoding the 5.8S ribosomal RNA subunit within the nuclear ribosomal cluster (ITS) and intron 2 of the glyceraldehyde-3-phosphate dehydrogenase gene (G3PD) were sequenced for isolates from PFD-affected sweet orange and KLA-affected Key limes collected in the United States (Florida), Brazil (S?o Paulo), Mexico, Belize, Costa Rica, and the Dominican Republic to determine if there are consistent genetic differences between PFD and KLA isolates over the geographic area where these diseases occur. Based on the sequence data, isolates clustered into two well-supported clades with little or no sequence variation among isolates within clades. One clade (PFD clade) contained PFD isolates from all countries sampled plus a few isolates from flowers of Key lime in Brazil. The other clade (KLA clade) contained KLA isolates from Key lime foliage from all countries sampled and one isolate from flowers of sweet orange in Mexico. In greenhouse inoculations with PFD and KLA isolates from Florida, isolates from both clades produced PFD symptoms on Orlando tangelo flowers, but KLA-clade isolates produced significantly less severe symptoms. PFD-clade isolates were not pathogenic to Key lime foliage, confirming previous studies. The differentiation of PFD and KLA isolates into two well-supported clades and the pathogenicity data indicate that PFD and KLA are caused by distinct phylogenetic lineages of C. acutatum that are also biologically distinct. PFD is a recently described disease (first reported in 1979) relative to KLA (first reported in 1912) and it had been proposed that strains causing PFD evolved from strains causing KLA eventually losing pathogenicity to Key lime foliage. We reject the hypothesis that PFD strains have diverged from KLA strains recently based on estimated divergence times of haplotypes and it appears that PFD and KLA strains have been dispersed throughout the Americas independently in association with each host.     

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.     

Infectivity analysis of a soybean isolate of Mungbean yellow mosaic India virus by agroinoculation

Yellow mosaic disease (YMD) of legumes endemic to South Asia are caused by begomoviruses transmitted by whiteflies. Based on molecular characterization, two distinct viruses – Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV) – were found previously to be the etiological agents of YMD in legumes. Here, host range studies with a soybean isolate of MYMIV (MYMIV-[Sb]) were carried out by both whitefly transmission and agroinoculation. MYMIV-[Sb] was similar to a cowpea isolate of MYMIV (MYMIV-[Cp]) in its ability to infect cowpea, thus differing from blackgram (MYMIV) and mungbean (MYMIV-[Mg]) isolates, which do not infect cowpea. Genomic analysis of DNA A and DNA B components of these MYMIV isolates show characteristic differences in complete DNA B nucleotide sequence correlating with host range differences.     

Fruit spot of sweet lime (<Emphasis Type="Italic">Citrus limetta</Emphasis>) caused by <Emphasis Type="Italic">Septoria</Emphasis> sp. in Peru

In 2002, a severe fruit spot of sweet lime (Citrus limetta) was observed in Piura and Lambayeque provinces in northern Peru. Affected fruits showed large oval and sunken lesions, often surrounded by chlorotic haloes. Septoria sp. was isolated from affected fruits. Sweet lime isolates showed larger pycnidia and pycnidiospores than those of Septoria spp. previously described on citrus. In addition, phylogenetic analysis of the ITS sequences clearly separated the sweet lime isolates from S. citri and S. citricola. Isolates were pathogenic to detached sweet lime fruits and the fungus was isolated from lesions on inoculated fruits.     

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.     

Inheritance and heritability of resistance to citrus leprosis

ABSTRACT The genetic inheritance of resistance to leprosis, the most important viral disease of citrus in Brazil, was characterized through the phenotypic assessment of 143 hybrids resulting from crosses between tangor 'Murcott' (Citrus sinensis x C. reticulata) and sweet orange 'Pêra' (C. sinensis), considered to be resistant and susceptible to the disease, respectively. All plants were grafted onto Rangpur lime (C. limonia) and inoculated with Citrus leprosis virus, cytoplasmic type through the infestation with viruliferous mites, Brevipalpus phoenicis. The experiments were arranged in a completely randomized block design with 10 replicates. Incidence and severity of the disease in leaves and stems as well as plant growth parameters (plant height and stem diameter) were recorded for 3 years after the infestation with the viruliferous mites. The average values of all variables were analyzed using principal component analysis, discriminant factorial analysis, estimation of the clonal repeatability coefficients, and frequency of the distributions of the average values for each measured variable. The principal component analysis resulted in the identification of at least two groups with resistance and susceptibility to leprosis, respectively. About 99% of all hybrids were correctly classified according to the discriminant factorial analysis. The broad-sense heritability coefficients for characteristics associated with incidence and severity of leprosis ranged from 0.88 to 0.96. The data suggest that the inheritance of resistance to leprosis may be controlled by only a few genes.     

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.