Incidence in Cyprus of citrus exocortis viroid and its mechanical transmission

A survey for citrus exocortis viroid (CEV) was conducted in commercial groves of the main citrus-producing areas of Cyprus during 1987–1990. Of 573 trees sampled, representing 25 species and varieties, 506 (88.3%) were found to be infected when indexed by grafting to seedlings of'Etrog' citron (Citrus medica) , selection Arizona 861. Levels of infection by CEV were lower in newer introductions than in introductions made before the 1940s. In greenhouse tests, CEV was readily transmitted from citron to healthy citron and gynura (Gynura aurantiaca) by knife-blade inoculation. Retransmission of CEV from infected gynura back to citron was successful.     

Host range and symptomatology of a graft-transmissible pathogen causing bud union crease of citrus on trifoliate rootstocks

A graft-transmissible pathogen causing bud union crease of Nagami kumquat SRA–153 on Troyer citrange was characterized for host range and symptomatology. Buds of Marsh grapefruit, Nules clementine, Eureka lemon and Pineapple sweet orange preinoculated with kumquat SRA–153 were propagated on citrange rootstocks. Some plants of Nules clementine and Eureka lemon had developed bud union crease six months after propagation, whereas all Marsh grapefruit and Pineapple sweet orange plants still showed normal bud union after one year. On indexing these preinoculated species, Nules clementine and Eureka lemon caused vein clearing in Pineapple sweet orange and Dweet tangor, chlorotic blotching in Dweet tangor and stem pitting in Etrog citron, whereas Marsh grapefruit and Pineapple sweet orange caused only chlorotic blotching in Dweet tangor and stem pitting in Etrog citron. Following shoot-tip grafting in vitro of kumquat SRA–153, kumquats 38–1 and 497–2 obtained from it caused chlorotic blotching in Dweet tangor and stem pitting in Etrog citron, but not vein clearing in Pineapple sweet orange and Dweet tangor or bud union crease when propagated on citrange. These results suggest the presence of at least two pathogens or pathogen strains in kumquat SRA–153 and the elimination of one of them after shoot-tip grafting in vitro or inoculation on Marsh grapefruit or Pineapple sweet orange. They also indicate that the pathogens in kumquat SRA–153 can be detected by indexing on Dweet tangor or Etrog citron.     

Separation of citrus viroids by shoot-tip grafting in vitro

Arizona 861-S1 citron ( Citrus media L.) infected with a severe exocortis isolate containing four citrus viroids was used as source of tissue for shoot-tip grafting in vitro. Out of 51 attempts, 25 successful grafts were obtained. Only 16 plants survived transplanting and of these 12 were viroid-free. The viroid profile of the other four plants showed fewer viroids than the original field source. The significance of these results, as compared with previous reports on the recovery of viroid-free plants, is discussed. The results show the usefulness of shoot-tip grafting in vitro as a tool to recover single viroid isolates from complex field sources.     

Indexing of Citrus Viroids by Imprint Hybridisation

A method based on the hybridisation of tissue imprints was developed for routine indexing of citrus viroids. For maximum sensitivity and reliability, the inoculation of Citrus medica (Etrog citron) as a viroid amplification host is required. Hybridisation against Digoxigenin-labelled RNA- or DNA-probes followed by detection of viroid-probe hybrids using anti-DIG-alkaline phosphate conjugate and the chemiluminescence substrate CSPD was suitable for the detection of all citrus viroids with the same sensitivity as other available methods. The overall process is extremely simple and allows quick analysis of large numbers of samples by easily trained personnel and minimum equipment.     

苹果锈果病(ASSD)类病毒的侵染性

 用机械接种的方法研究了苹果锈果病类病毒(ASSV)的侵染性.在嫁接时将接种原滴于砧木和接穗的切面上,用针刺切面,然后观察症状表现,所有接穗都取自同一株健康“国光”.用半提纯的ASSD—RNA—1和ASSD—RNA—2接种的,成活的3株中有2株发病;用提纯的ASSD—RNA—1和ASSD—RNA—2接种的,成活的3株中有1株发病;用病组织的澄清汁液接种的,成活的4株中有1株发病;用缓冲液模拟接种的健康对照成活的5株均未发病.发病苗木的症状为叶片卷曲,随后叶片从叶柄处断裂.从发病的,不发病的及健康对照的苗木茎部提取核酸,用双向及两次聚丙烯酰胺凝胶电泳分析,表现症状的4株均测出了ASSD—RNA—1,未发病的及用缓冲液模拟接种的健康对照均未检测出类病毒核酸带.结果表明ASSD—RNA—1可通过机械传播,为苹果锈果病的类病毒病原提供了又一次证据,也表明ASSD—RNA—1代表了能独立复制的苹果锈病类病毒.     

Peach red marbling and peach sooty ringspot, two new virus-like degenerative diseases of Prunus

More than 600 Prunus samples were examined by using a nonradioactive digoxigenin-labelled RNA probe specific for hop stunt viroid (HSVd). Prunus salicina and Prunus armeniaca appeared to be better hosts than Prunus persica . The weak viroid concentration in flowers and young leaves of peach trees growing in the field did not permit its detection in such samples. The diagnosis was more reliable (about 85%) with bark and leaves aged 4 months and more, from regrowths of GF 305 peach seedlings inoculated and kept in the greenhouse. Detection of HSVd in leaves and bark of apricot and Japanese plum plants aged 3 months or more also proved reliable (about 80% and 90%, respectively). HSVd could be transmitted in apricot, peach and plum nucleic acid preparations to GF 305 peach seedlings by repeated stem slashing, and to cherries ( Prunus avium and Prunus serrulata ) by approach grafting with an infected P. salicina source. The viroid was eliminated from 18% of the clones obtained after thermotherapy.
In the course of this study, 25 selected Prunus accessions suspected to be infected by unusual diseases were analysed by hybridization with a HSVd-specific probe and by indexing on GF 305 peach seedlings in the greenhouse. Fifteen of these accessions were found to be infected by HSVd, 19 induced reddish marbling, and four induced small blackish spots on the leaves aged about 4 months. Repeated assays showed that these foliar symptoms were not caused by the viroid. Peach red marbling (PRMa) has not been associated with any known virus and seems to be caused by an infectious agent not yet described. That could also be the case with the agent of peach sooty ringspot (PSRS). PRMa and PSRS symptoms were reproduced by grafting and indexing, and their causal agents eliminated by thermotherapy in a significant fraction of the treated plants. They behave like viral agents and can infect the different Prunus species studied.     

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。     

我国发现由甘薯褪绿矮化病毒和甘薯羽状斑驳病毒协生共侵染引起的甘薯病毒病害

甘薯病毒病害(Sweet potato virus disease,SPVD)是由毛形病毒属(Crinivirus)的甘薯褪绿矮化病毒(Sweet potato chlorotic stunt virus,SPCSV)和马铃薯Y病毒属(Potyvirus)的甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFMV)协生共侵染甘薯引起的病毒病害[1].     

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.     

A new disease of greenhouse tomatoes in Israel caused by a distinct strain ofTomato apical stunt viroid (TASVd)

Using the sequential PAGE method for detection of small circular RNA molecules we isolated a viroid from greenhouse-grown tomato plants exhibiting severe stunting in Israel. The viroid was transmitted to tomato and to several other solanaceous plants by graft and mechanical inoculation, but only tomato plants showed symptoms of disease. Cloning and sequencing revealed that the viroid RNA is composed of 363 nucleotides, has 92% identity with the type strain (Ivory Coast strain) ofTomato apical stunt viroid (TASVd) and 99% identity with the Indonesian strain of this viroid. The experimental host range of TASVd-Is differs significantly from that of the type strain of TASVd. The possible epidemiological consequences leading to TASVd spread in geographically distant areas are discussed. http://www.phytoparasitica.org posting Sept. 18, 2002. Corresponding author     

Citrus viroid II variants associated with ‘Gummy Bark’ disease

A viroid etiology for citrus gummy bark (CGB) disease of sweet orange is supported by the similarity of symptom expression to cachexia disease of mandarins and tangelos caused by the hop stunt viroid (HSVd) related citrus viroid II (CVd-II), as well as the detection of CVd-II variants in CGB infected Washington navel and Dörtyol sweet orange, a Turkish cultivar. A survey was made of 67 clones of CVd-II related variants recovered from severe CGB symptomatic and non-symptomatic trees of the same cultivars growing in close proximity. Only CVd-IIa, a non-cachexia inducing variant, was found in non-symptomatic Washington navel trees and no CVd-II variants were recovered from the Dörtyol control. CGB infected sources contained a number of CVd-II related variants with the predominant species detected closely related to CVd-IIc, a known cachexia inducing viroid. Biological activity of representactive variants from CGB sources was determined by transmission to citron (Citrus medica) as well as by bioassay on the indexing host for cachexia, Parson's Special mandarin (Citrus reticulata).     

柑桔裂皮病类病毒分子生物学研究进展

本文就柑桔裂皮病类病毒(citrus exocortis viroid,CEVd)的分类地位,分子结构及其生物学鉴定、聚丙烯酰胺凝胶电泳分析和分子杂交、分子生物学检测等方法进行了综述。     

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.     

Sensitivity of Crinipellis perniciosa to two triazole fungicides in vitro and their effect on development of the fungus in cocoa

Two triazole fungicides, hexaconazole and triadimenol, were evaluated for their effects on the growth of Crinipellis perniciosa in vitro, and for their ability to prevent broom formation on cocoa seedlings. Amounts of both fungicides required to reduce fungal growth, germ tube extension and basidiospore germination by 50% were found respectively to be less than 1, 10-150 and more than 200 mg/l. Reductions in germ tube length were associated with slower rates of cell growth, whereas growth of dikaryotic mycelium was reduced due to shorter cell lengths. Isolates of C. perniciosa showed different sensitivities to the fungicides, variation between collections from different localities being greater than between collections from the same locality. Hexaconazole gave good control of the disease applied as a spray to cocoa seedlings both before and after inoculation. Triadimenol showed good activity when used as a preinoculation soil drench and in some treatments where plants became infected no necrosis of host tissue was observed. Activity was greatly reduced by drench treatments applied 10 days after inoculation, and most infected plants formed necrotic cankers.     

Diagnostic techniques for viroids

The fast growth of the human population forces us to produce more food, but higher crop production also leads to the fast spread of diseases. Plant pathology deploys a wide range of methods that do not provide an adequate solution to all disease losses. In the case of viroids, therapeutic means of control are not available; therefore control strategies are more focused on the development of reliable detection methods to quickly exclude the infected plant material. Although viroids are the smallest and simplest plant pathogens, their identification and detection is not straightforward. Each viroid–host combination is specific, and for reliable identification, all steps from sampling to final detection must be performed accurately. In this review, several methods for viroid detection in various host plants are discussed, including their advantages and disadvantages. Even though relatively new molecular methods enable fast and sensitive detection of viroids, a combination of different methods gives the most reliable identification. Techniques based on nucleic acids may be the future for viroid detection but they still cannot replace biological indexing, which is usually essential in epidemiological and aetiological studies.     

Scanning electron microscopy of the interactions betweenerwinia herbicola pv.gypsophilae andgypsophila paniculata

Ultrastructural studies of interactions betweenErwinia herbicola pv.gypsophilae and gypsophila plants showed that the bacterial pathogen multiplied rapidly and colonized gypsophila cuttings within 2 days. Fourteen days after inoculation, the plant tissue turned tumorigenic and a gall formed. Scanning electron microscopy showedE. gypsophilae cells densely occupying the plant xylem elements. Bacteria were found in association with fibrils which might be used by bacterial cells for attachment to plant cells. Substantial numbers of fibrils were also clearly seen on calli cell surfaces.     

Local and Systemic Induced Resistance to the Root-Knot Nematode in Tomato by DL-beta-Amino-n-Butyric Acid

ABSTRACT Chemical inducers of pathogenesis-related proteins and plant resistance were applied to tomato plants, with the aim of inducing resistance to the root-knot nematode Meloidogyne javanica. Relative to control plants, foliar spray and soil-drenching with dl-beta-amino-n-butyric acid (BABA) reduced root-galling 7 days after inoculation, as well as the number of eggs 30 days after inoculation. Other chemicals (alpha- and gamma-amino-n-butyric acid, jasmonic acid, methyl jasmonate, and salicylic acid) were either phytotoxic to tomato plants or did not improve control of root-knot nematodes. Fewer second-stage juveniles invaded BABA-treated tomato roots, and root-galling indices were lower than in control tomato plants. Resistance phenomena in seedlings lasted at least 5 days after spraying with BABA. Nematodes invading the roots of BABA-treated seedlings induced small, vacuolate giant cells. Postinfection treatment of tomato plants with BABA inhibited nematode development. It is speculated that after BABA application tomato roots become less attractive to root-knot nematodes, physically harder to invade, or some substance(s) inhibiting nematode or nematode feeding-site development is produced in roots.     

Effect of method of inoculation, inoculum density and seedling age at inoculation on the expression of resistance of cocoa (Theobroma cacao L.) to Verticillium dahliae Kleb.

Soil drench and stem puncture inoculation were compared as methods for selecting cocoa cultivars with resistance to Verticillium dahliae. Disease progress was more rapid and induced symptoms were more severe following stem puncture and, under glasshouse conditions, differences between cultivars were detected 15 days after inoculation. Moreover, using stem puncture, inoculum densities of 10⁴ conidia/ml were sufficient to differentiate resistant and susceptible cultivars, whereas with the soil drench method, inoculum densities of 10⁷ conidia/ml were necessary. Although a substantially higher proportion of plants were affected by stem puncture inoculation, the resistance of cultivar Pound-7 remained effective at high inoculum densities of 10⁸ conidia/ml. With either method, older seedlings were more susceptible to V. dahliae than younger ones. However, with stem puncture, 15-day-old seedlings were sufficiently susceptible for a valid disease assessment. In contrast, with soil inoculation, 60-day-old plants were required. In a nursery trial with 15-day-old seedlings, seven cocoa genotypes previously selected as resistant, moderately resistant or susceptible to Verticillium dahliae , on the basis of root inoculation, were ranked in the same order when stem punctured. Stem puncture inoculation of young seedlings is cost-effective in terms of time and space, and is therefore recommended for screening of cocoa for wilt resistance, especially in large-scale breeding programmes.