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.     

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.     

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.     

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.     

柑桔体细胞杂种对柑桔脚腐病的抗病性研究

 三七[Panax notoginseng(Burk) F.H.Chen,1945]是我国的名贵药材之一,与三七有关的各种病害的研究报道较多,但根结线虫危害三七,引起三七根结线虫病尚未见报道。     

Morphological and molecular characterization of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop in Brazil

Brazilian isolates of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop were examined for colony colour, mycelial growth, benomyl-resistance, pathogenicity, and genetic variability by random amplified polymorphic DNA (RAPD) analysis. All isolates were obtained from flowers and persistent calyxes from different citrus hosts from Sao Paulo, Brazil. DNA polymorphisms detected after amplification with random 10-mer primers were used to classify the isolates into two groups. Group I isolates grew rapidly on potato-dextrose agar (PDA) and were sensitive to benomyl, and group II isolates grew slowly on PDA and were benomyl-resistant. Colletotrichum acutatum was analyzed by RAPD and had high genetic similarity with group II isolates of Colletotrichum from citrus. Probably, the group I is C. gloeosporioides and group II is C. acutatum.     

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.     

摩洛哥脐血橙植株上一种机械传播性病毒的鉴定

 本文采用叶片接种法和枝条接种法对25个柑桔种类体细胞杂种或品种进行了抗脚腐病鉴定,结果表明:体细胞杂种中枳+酸橙、枳+甜橙为高抗类型;酸橙+兰普来檬、枳+印度酸桔;蚝壳刺+哈姆甜橙、酸橙+澳洲指桔为抗性;酸橙+印度酸桔为中抗。建立了叶片接种法的抗病性分级标准。初步分析了柑桔体细胞杂种的抗性规律。     

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.     

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。     

Use of phages for identifying the citrus canker bacterium Xanthomonas campestris pv. citri in Taiwan

Phages CP115 and CP122, which were isolated from canker lesions on grapefruit and Liucheng sweet orange, respectively, showed a high degree of specificity with respect to lysis of test bacterial strains. When used jointly, they lysed 135 (97·8%) out of 138 Xanthomonas campestris pv. citri strains isolated from the canker lesions on leaves, twigs, and fruits of various citrus species, cultivars, and hybrids grown throughout Taiwan, but they did not lyse other X. campestris pathovars and other phytopathogenic bacteria, nor other bacteria isolated from soil, clinical or environmental samples. Of 252 CP115/CP122-sensitive and 78 CP115/CP122-resistant bacterial strains with colony characteristics typical of or similar to those of X. campestris pv. citri , isolated from canker lesions of various citrus plants in diverse growing regions in Taiwan, 250 (99·2%) and 76 (97·4%) strains were pathogenic and non-pathogenic, respectively, when inoculated into Liucheng sweet orange or Mexican lime. Thus, phages CP115 and CP122, when used jointly, appear to be applicable for identifying X. campestris pv. citri in Taiwan.     

Infection of citrus pollen grains by Colletotrichum acutatum

Postbloom fruit drop (PFD), an important disease caused by Colletotrichum spp., affects citrus yields in Brazil. PFD is characterised by the presence of necrotic lesions on the petals and stigmas of citrus flowers and by the subsequent abscission of young fruit. PFD epidemics have high disease progress rates, which is unusual for a pathogen that produces acervuli and is dispersed by rain. It is possible that other dispersal agents, such as insects and pollen, are involved in the spread of this disease. The objective of this work was to test whether citrus pollen grains can be colonised by Colletotrichum acutatum. Studies using light and electron microscopy showed that the pollen of Citrus sinensis can be infected by C. acutatum. This pathogen can penetrate and colonise citrus pollen grains 24 h after inoculation with the pathogen. The germ tube of conidia either penetrates the pollen sporodermis directly or passes through pollen germ pores. A single hypha can colonise more than one pollen grain. On the surface of the stigma, conidium formation can be observed. This study shows that Citrus sinensis pollen may, in fact, play a role in the spread of C. acutatum in citrus orchards.     

Characterization of Colletotrichum acutatum Isolates Causing Anthracnose of Almond and Peach in California

ABSTRACT The causal organism responsible for the recent outbreak of almond and peach anthracnose in California was identified and characterized as Colletotrichum acutatum. Isolates of C. acutatum from almond were found to be similar to California strawberry isolates and South Carolina peach and apple isolates of C. acutatum based on conidial morphology, temperature relationships, fungicide sensitivity, and polymerase chain reaction (PCR) methods using DNA species-specific primers. On almond, blossoms and immature or mature fruit were affected by the disease, causing direct losses of crop. On peach, the disease was observed only on mature fruit. Pathogenicity of almond and peach isolates of C. acutatum was demonstrated on wound- and nonwound-inoculated almond or peach fruit by fulfilling Koch's postulates. Conidial morphology of isolates was variable, depending on the medium or substrate used to culture the isolates. Isolates of C. acutatum from strawberry, almond, and peach were grouped together based on a similar response to temperature, with an optimal growth rate at 25 degrees C (generally less than 10 mm/day), whereas isolates of C. gloeosporioides from citrus and papaya had an optimal growth rate at 30 degrees C (generally greater than 10 mm/day). In fungicide disk assays, isolates of C. acutatum from strawberry, peach, and apple, as well as almond and peach isolates from California, were less sensitive to benomyl at 300, 600, or 1,200 mug/ml. In contrast, C. gloeosporioides isolates from citrus and papaya were very sensitive to benomyl at all concentrations evaluated. All isolates of both species were sensitive to captan (300, 600, or 1,200 mug/ml). Oligonucleotide primers were synthesized for C. acutatum, C. fragariae, or C. gloeosporioides using published DNA sequences from the internal transcribed spacer 1 region of ribosomal DNA. Thirty-two Colletotrichum isolates from almond fruit produced DNA products with a C. acutatum primer (CaInt-2) that matched products and approximate molecular weight of known C. acutatum isolates. No PCR products were produced with primers for C. gloeosporioides or C. fragariae. Isolates from citrus and papaya produced DNA products only with primers from C. gloeosporioides or C. fragariae. Thus, worldwide, anthracnose of almonds may be caused by either C. gloeosporioides, as previously reported, or by C. acutatum, as indicated in this study.     

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.     

Identification of colletotrichum species responsible for anthracnose and root necrosis of strawberry in Israel

ABSTRACT Strawberry anthracnose was observed for the first time in Israel in 1995. The disease reached epidemic proportions in Israeli nurseries and production fields in 1995 and 1996. Using morphological and cultural characteristics, the species responsible for anthracnose was identified as Colletotrichum acutatum. A reliable semi-selective medium, amended with iprodione and lactic acid, was used to isolate the fungus from infected tissues. In addition, C. acutatum was subsequently isolated from necrotic roots of stunted, chlorotic plants that exhibited no symptoms of anthracnose. High levels of the pathogen from naturally infested field soil and perlite growth substrate were quantified from the rhizosphere of diseased plants on the iprodione-amended medium. Both foliar- and rootinfecting isolates were equally pathogenic to strawberry, causing 95 to 100% plant mortality, when inoculated on roots and foliage. In complementation (heterokaryon) tests using nitrate nonutilizing mutants, 113 out of 115 isolates from different plant parts and locations belonged to a single vegetative compatibility group. Arbitrarily primed polymerase chain reaction of genomic DNA using four repetitive-motif primers produced nearly uniform amplified DNA banding patterns for 141 of the Israeli strawberry isolates from different sites, plots, plant tissues, and cultivars. When compared to reference isolates from the US, these band patterns suggested that a single introduction of C. acutatum was responsible for strawberry anthracnose on foliage and necrosis of roots in Israel.     

Citrus Black Rot is Caused by Phylogenetically Distinct Lineages of Alternaria alternata

ABSTRACT Phylogenetic analysis revealed that isolates of Alternaria alternata causing black rot of citrus were associated with six well-supported evolutionary lineages. Isolates recovered from brown spot lesions on Minneola tangelo, leaf spot lesions on rough lemon, and healthy citrus tissue and noncitrus hosts were related closely to isolates from black-rotted fruit. Phylogenies estimated independently from DNA sequence data from an endopolygalacturonase gene (endoPG) and two anonymous regions of the genome (OPA1-3 and OPA2-1) had similar topologies, and phylogenetic analysis was performed on the combined data set. In the combined phylogeny, isolates from diverse ecological niches on citrus and noncitrus hosts were distributed in eight clades. Isolates from all lineages, regardless of ecological or host association, caused black rot in fruit inoculation assays, demonstrating that small-spored Alternaria isolates associated with different ecological niches on citrus and other plant hosts are potential black rot pathogens. These data also indicated that the fungi associated with black-rotted fruit do not form a natural evolutionary group distinct from other Alternaria pathogens and saprophytes associated with citrus. The use of the name A. citri to describe fungi associated with citrus black rot is not justified and it is proposed that citrus black rot fungi be referred to as A. alternata.     

6种柑橘类植物对柑橘衰退病毒分离株TR-L514变异的影响

 柑橘衰退病毒(Citrus tristeza virus,CTV)存在复杂的株系分化现象,运用弱毒株交叉保护防治柑橘衰退病时需了解不同类型柑橘对CTV构成的影响。作者将CTV分离株TR-L514嫁接接种到6类柑橘植物上获得了18个亚分离株,并对这18个亚分离株和TR-L514进行了指示植物鉴定,p25基因的限制性片段长度多态性(RFLP)和单链构象多态性(SSCP)分析,p23基因的序列比较。结果表明,CTV株系对不同柑橘类植株的适应性存在差异。TR-L514嫁接到不同柑橘类植株其构成会发生变化,在甜橙上可以同时检测出p25//HinfⅠ RFLP第1和6组群,并具有比在其它4类柑橘上更为复杂的SSCP谱型构成,因此甜橙可能更适宜于CTV的增殖。TR-L514及18个亚分离株的p23基因序列差异可能与不同类柑橘植株的适应性有关。     

Colletotrichum gloeosporioides, a new causal agent of citrus post-bloom fruit drop

Citrus post-bloom fruit drop (caused by Colletotrichum acutatum) frequently occurs in the southwestern region of São Paulo State, Brazil. A survey of Colletotrichum isolates associated with symptoms of post-bloom fruit drop in São Paulo State showed C. gloeosporioides in addition to C. acutatum. The objectives of this study were to confirm the identification of C. gloeosporioides isolated from symptomatic citrus flowers, to test the pathogenicity of C. gloeosporioides isolates, to compare the development of disease caused by C. gloeosporioides and C. acutatum, and to determine the frequency of C. gloeosporioides in a sample of isolates obtained from symptomatic flowers in different regions of São Paulo State. Through the use of species-specific primers by PCR, 17.3% of 139 isolates were C. gloeosporioides, and the remaining 82.7% were C. acutatum. The pathogenicity tests, carried out in 3-year old potted plants of sweet oranges indicated that both species caused typical symptoms of the disease including blossom blight and persistent calyces. Incubation periods (3.5 and 3.9 days, respectively, for C. acutatum and C. gloeosporioides) and fruit sets (6.7 and 8.5%, respectively for C. acutatum and C. gloeosporioides) were similar for both species. The incidences of blossom blight and persistent calyces were higher on plants inoculated with C. acutatum than in those inoculated with C. gloeosporioides. Conidial germination was similar for both species under different temperatures and wetness periods. Under optimal conditions, appressorium formation and melanisation were higher for C. gloeosporioides than for C. acutatum. These results indicated that Colletotrichum gloeosporioides is a new causal agent of post-bloom fruit drop.