Sampling conditions for reliable routine detection by enzyme-linked immunosorbent assay of three ilarviruses in fruit trees

Enzyme-linked immunosorbent assay (ELISA) was used to test plum trees for prune dwarf (PDV), Prunus necrotic ringspot (NRSV) and apple mosaic (ApMV) viruses, cherry trees for PDV and NRSV, and apple trees for ApMV. Optimum conditions were determined for sampling in large-scale surveys for these viruses. All three viruses were detected throughout the growing season in individual samples of young leaves, or twigs with newly formed buds. However, when single infected leaves were combined with different numbers of healthy leaves, tests were most successful for all three viruses early in the growing season. PDV was detected in 1/40 (infected/total leaves) cherry leaves in April and May and 1/40 plum leaves until July, whereas NRSV was detected in 1/20 cherry leaves until July and 1/20 plum leaves until May. ApMV was detected in 1/20 apple or plum leaves until June but was detected less readily in mature leaves after June than either NRSV or PDV. There was no evidence of uneven distribution of virus infection in the trees. The viruses were detected in leaf samples kept for 8 wk at 3°C but freezing was less reliable for storage especially with ApMV. ApMV was detected in tests on plants held for several weeks at 25°C, and PDV and NRSV in plants held at 30°C.     

Strains of Prunus necrotic ringspot virus in hop (Humulus lupulus L.)

Purified preparations of Prunus necrotic ringspot virus (NRSV) from hop plants formed two light-scattering zones when centrifuged in sucrose density gradients; the upper and lower zones contained particles 25 mμ and 31 mμ in diameter respectively whose sedimentation coefficients were 79 S and 107 S. NSRV isolates from hop were of two distinct serological types: ‘A’ strains, serologically very closely related to NRSV isolates from apple; and ‘C’ strains more nearly related to NRSV from cherry. The variety Fuggle is tolerant to hop mosaic (not related to NRSV) and different selections of apparently healthy female plants usually contained A strains; but C strains were usually isolated from nettlehead-diseased plants. Either A or C strains occurred in male plants grown with the hop-mosaic tolerant varieties. In mosaic-sensitive varieties (Goldings and Bramlings) apparently healthy female plants tested were usually infected with C strains; either A or C types occurred in mosaic-sensitive male plants. NRSV was not detected in the seventy-four hop seedlings obtained from virus-infected plants. Some varieties developed nettlehead when infected with NRSV (A) or (C) + the hop form of arabis mosaic virus, but not with NRSV (A) or (C) alone. Others developed nettlehead when infected with arabis mosaic virus + NRSV (C) but not with arabis mosaic + NRSV (A). A and C strains can multiply together in the same hop plant. There is evidence of partial antagonism, however, and the fluctuating behaviour of the nettlehead syndrome probably reflects changes in the relative concentration of the two serotypes.     

Identification of viruses isolated from plum trees affected by decline, line-pattern and ringspot diseases

Viruses obtained from plum trees infected with either decline, line-pattern or ringspot diseases were characterized and identified. All the viruses were serologically related to Prunus necrotic ringspot virus (NRSV); those from trees with decline or ringspot were serologically indistinguishable from cherry strains of NRSV but differed in pathogenicity, whereas the virus from trees with line pattern was closely related to apple mosaic virus. When returned from herbaceous hosts to Prunus, line-pattern and ringspot isolates reproduced symptoms characteristic of the diseased sources. One virus isolate from trees with decline diminished the vigour of young plum trees. Comparison with other investigations shows that at least two unrelated viruses cause plum line-pattern disease in America and Europe.     

Use of protein A to improve sensitisation of latex particles with antibodies to plant viruses

Protein A-coated latex (PAL) was compared with uncoated latex (L) for sensitisation with antibodies to five plant viruses: apple mosaic virus (ApMV), arabis mosaic virus (AMV), plum pox virus (PPV), potato virus Y ordinary strain (PVY°) and prunus necrotic ringspot virus (NRS V). A range of globulin concentrations was used with each antiserum and detection end points determined in serial dilutions of infective sap. When sensitised with antibodies to ApMV, PAL detected ApMV readily, whereas L did not. When sensitized with antibodies to PVY° and AMV, PAL gave higher detection end points than L. However, PAL gave little increase in sensitivity with the antisera to PPV and NRSV. Non-specific aggregation of latex, which sometimes occurred in very dilute sap with PAL, could be dispersed by adding 0.02% Tween-20 to the extraction buffer. Globulins of PVY° and AMV could be used at higher dilutions with PAL than with L, giving a saving in antiserum. Both types of latex sensitised with PVY° antibody globulins readily detected the tobacco veinal necrosis and C strains of this virus.     

The serological relationships and some other properties of isolates of broad bean wilt virus from faba bean and pea in China

An isolate (N15) of broad bean wilt virus (BB W V) from faba bean in China was compared with some other isolates and strains including the nasturtium ringspot strain (NRSV, BBWV serotype I), parsley virus 3 (PV3, serotype I) and BBWV isolate PV131 (serotype II). In host range studies, N15 infected 12 of 14 species, including soybean and spinach. It was purified from Chenopodium quinoa and pea by a method that yielded up to 8mg/100g tissue. By the same method, NRSV yielded up to 4mg/100 g. Purified preparations of N15 and NRSV contained isometric particles c. 26 nm in diameter which sedimented as three components, N15 at 62, 93 and 117 S, and NRSV at 60, 91 and 116 S. In immunodiffusion tests using antisera to N15 and NRSV, N15 was distinguishable from NRSV but indistinguishable from PV131. In ISEM tests, many more particles of N15 and NRSV were trapped by homologous than by heterologous antiserum; in decoration tests, much antibody attached to homologous particles but none to heterologous particles. In DAS ELISA using N15 antiserum, N15 and six other Chinese faba bean or pea isolates, and a Chinese spinach isolate, were readily detected and were indistinguishable from each other and from PV131; unlike NRSV and PV3, none of the Chinese isolates, nor PV131, was detected using NRSV antiserum. These results indicate that the Chinese isolates belong to BBWV serotype II group.     

Apple mosaic virus isolated from hop plants in Japan

A strain of apple mosaic virus was isolated from hop plants in Japan. The virus was purified from young hop plants and back-inoculated to virus-free hop plants obtained by meristem tip culture. Inoculated plants developed chlorotic spots, ringspots and a band pattern accompanied by necrosis in the inoculated and systemically infected leaves. Shoot tips of infected plants sometimes became necrotic and these symptoms resembled those of a ring- and band-pattern mosaiclike disease prevalent in hop gardens in Japan. Since apple mosaic virus was recovered from infected plants, it is likely that the virus was the causal agent of this disease. Agar gel double diffusion tests and ELISA showed the hop virus to be serologically closely related to apple mosaic virus (ApMV), and distantly related to prunus necrotic ringspot virus (PNRSV). The virus had a narrow host range, and infected only cucumber of 18 species of Cucurbitaceae, Chenopodiaceae, Leguminosae or Solanaceae inoculated. It produced chlorotic spots on the inoculated cotyledons of cucumber, but no systemic infection. By contrast, ApMV from apple and PNRSV from peach had wide host ranges and infected cucumber plants systemically.     

Mechanical transmission of Apple mosaic virus in Australian hop (Humulus lupulus) gardens

The transmission of Apple mosaic virus (ApMV; hop, H and intermediate, I serotypes) in Australian hop cultivars was assessed in glasshouse and field trials. Under field conditions, the rate of ApMV transmission was halved when contact between neighboring plants was prevented by early season applications of paraquat to restrict basal shoot growth. However, in a separate field trial the presence of root grafts between hop plants, which may contribute to virus transmission, was also suggested. In glasshouse trials, ApMV was transmitted successfully to hop by the mechanical inoculation of infective sap, simulated pruning, foliar contact, and root grafting, but not by root contact. The rate of mechanical transmission of ApMV to the hop cultivar ‘Victoria’ was greater than to other hop cultivars commonly grown in Australia. However, success of mechanical transmission of ApMV also appeared to be influenced by the cultivar from which inoculum was obtained. ApMV was detected throughout the year in all tissues, in chronically infected field grown plants of cultivar ‘Victoria’, suggesting a uniform virus distribution. The reliability of ApMV detection by serology did not decline in ‘Victoria’ plants later in the growing season as occurred in other cultivars.     

Development of multiplex RT-PCR assay for simultaneous detection of four viruses infecting apple (Malus domestica)

The major viruses infecting apple cultivars throughout the world including India are apple mosaic virus (ApMV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV), and recently, a new virus, apple necrotic mosaic virus (ApNMV), was reported from mosaic-infected apple cultivars in India. The aim of this study was to detect the ApNMV virus along with the other three viruses (ApMV, ASPV and ASGV) simultaneously by multiplex RT-PCR. Four primer-pair-produced amplicons of 670, 550, 350 and 210 bp corresponding to ApNMV, ApMV, ASPV and ASGV, respectively, were found to be specific for these viruses when tested individually. The annealing temperature (55°C), primer concentration (0·8 µl) and other components of the master mix were standardized for the development of one-step m-RT-PCR assay. The m-RT-PCR protocol developed was further validated with 30 samples from seven symptomatic or asymptomatic apple cultivars, which revealed the presence of more than one virus in these cultivars. Most of the viruses were found to be present either alone or in mixed infection; however, ASPV was more common in tested cultivars. An easy, cost-effective and rapid multiplex RT-RCR protocol was developed to detect the four viruses, which infect apple plants either in individually or together in the field. This assay will help in the surveying and indexing of apple germplasm and the distribution of all four viruses in the apple growing regions of India.     

Arabis mosaic and Prunus necrotic ringspot viruses in hop (Humulus lupulus L.)

Purified virus preparations made from nettlehead-diseased hop plants, or from Chenopodium quinoa, to which the virus was transmitted by inoculation of sap, contained polyhedral virus particles of 30 mμ diameter which were identified serologically as arabis mosaic virus (AMV). There were serological differences between AMV isolates from hop and from strawberry, and also differences in host range and in symptoms caused in C. quinoa and C. amaranticolor. AMV was always associated with nettlehead disease. The nematode Xiphinema diversicaudatum occurred in small numbers in most hop gardens, but was numerous where nettlehead disease was spreading rapidly. Preparations from nettlehead-affected hops also contained a second virus, serologically related to Prunus necrotic ringspot virus (NRSV), in mild and virulent forms which infected the same range of test plants but showed some serological differences. Mild isolates did not protect C. quinoa plants against infection by virulent isolates. Hop seedlings inoculated with virulent isolates of NRSV developed symptoms indistinguishable from those of split leaf blotch disease. Latent infection with NRSV was prevalent in symptomless hop plants. Nettlehead disease is apparently associated with dual infection of AMV and virulent isolates of NRSV. An unnamed virus with rod-shaped particles 650 mμ long was common in hop and was transmitted by inoculation of sap to herbaceous plants. Cucumber mosaic virus was obtained from a single plant of Humulus scandens Merr.     

A yellow mosaic disease of horse chestnut (Aesculus spp.) caused by apple mosaic virus

A severe foliar yellow mosaic disease was observed in horse chestnut trees (Aesculus carnea and A. hippocastanum). Reactions in woody indicator plants grafted with diseased horse chestnut suggested the presence of an ilarvirus. Virus isolates obtained by mechanical inoculation of herbaceous test plants reacted with antisera to apple mosaic virus but not with antisera to its serotype prunus necrotic ringspot virus, or to prune dwarf virus. Yellow mosaic was induced in horse chestnut seedlings grafted with tissues from herbaceous hosts infected with horse chestnut isolates or with the European plum line pattern isolate of apple mosaic virus. Virus was detected by enzyme-linked immunosorbent assay (ELISA) in embryo and endosperm of immature seed from infected trees but not in mature seed, or progeny seedlings. Strawberry latent ringspot virus was detected in one of six A. hippocastanum trees with a leaf vein yellows disease.     

THE LINE-PATTERN VIRUS DISEASE OF PLUMS

The disease, now usually called line-pattern of plum, has been described under many names in most countries where plums are grown extensively.
Naturally infected trees show widely differing Symptoms; this has two causes: (1) different varieties react differently to the same isolate of the virus, and (2) different isolates cause different symptoms in the same variety. Because the virus occurs in strains with different pathogenicities, the choice of indicator varieties is important when selecting virus-free material by transmission tests. Peach seedlings and the mazzard clone, F 12/1, were the most sensitive types found.
The line-pattern virus does not become fully systemic in some varieties of plum. In this and other respects, it resembles the viruses that cause apple mosaic; three isolates obtained from plum and two from apple produced similar Symptoms in peach and apple. It is therefore suggested that plum line-pattern and apple mosaic viruses are caused by strains of one virus.     

Cross-immunity and antibody responses to different immobilisation serotypes of Ichthyophthirius multifiliis

Vaccination of channel catfish with either of two serotypes of the parasitic ciliate Ichthyophthirius multifiliis conferred protection against challenge infection by either serotype. Fish were vaccinated by intracoelomic injection with live theronts of isolate G5 (serotype D) or isolate G12 (a new serotype), which express different surface immobilisation antigens. Vaccination with live G12 theronts conferred complete protection against subsequent challenge by both serotypes while vaccination with G5 theronts elicited only partial protection against both serotypes. Vaccination with trophont lysates did not protect against challenge infection. Sera from vaccinated fish were tested in immobilisation assays, ELISAs, and Western blots. Serum antibodies recognised only immobilisation antigens of the serotype used for vaccination in immobilisation assays or on Western blots. No antigens common to both serotypes were identified by Western blots. In contrast, serum antibodies bound antigens in cell lysates from both serotypes by ELISA, demonstrating that antibodies recognising both serotypes are produced in response to infection, which presumably confer observed cross-serotype protection.     

Versuche zur Ausarbeitung einer Laborprüfmethode zwecks Feststellung der Knollenresistenz gegenüber Phytophthora infestans

Beim routinemäßigen Nachweis mechanisch übertragbarer Viren in Bäumen des Kern‐ und Steinobstes kann die Probeentnahme das Testergebnis u. U. nachhaltig beeinflussen. Die Kenntnis der Verteilung dieser Erreger in der Baumkrone ist deshalb von entscheidender Bedeutung. Folgende Viren wurden in die Untersuchungen einbezogen: Chlorotisches Apfelblattfleckungs‐Virus (apple chlorotic leaf spot virus, CLSV), Apfelstammfurchungs‐Virus (apple stem grooving virus, SGV) und Apfelmosaik‐Virus (apple mosic virus, ApMV) (Kernobst) bzw. Nekrotisches und Chlorotisches Kirschenringflecken‐Virus (Prunus necrotic ringspot virus, PNRV; prune dwarf virus, PDV), ApMV, CLSV, Scharka‐Virus der Pflaume (plum pox virus, PPV), Petunia asteroid mosaic virus (PAMV) und Kirschenblattroll‐Virus (cherry leaf roll virus, CLRV) (Steinobst). In der Regel kam der ELISA, nur in Einzelfällen der Latextest, zur Anwendung. Die genannten Viren lassen sich hinsichtlich der Verteilung in der holzigen Wirtspflanze 3 Gruppen zuordnen:

• Viren mit systemischer Verteilung: CLSV, SGV, PNRV, PDV, PPV in hochanfälligen Pflaumensorten und Pfirsich

• Viren mit teilsystemischer Verteilung: ApMV, PPV in weniger anfälligen Pflaumensorten

• Viren mit sporadischer Verteilung: PAMV, CLRV

     

Effect of viruses on agronomic and brewing characteristics of four hop (Humulus lupulus) cultivars in Australia

The effect of Hop latent virus (HpLV), Hop mosaic virus (HpMV), and Prunus necrotic ring spot virus [PNRSV (apple, A, and intermediate, I, serotypes)], on the survival of softwood cuttings, the vigour (height) of early season growth, cone yield, and the levels of brewing organic acids in mature plants, was assessed in four hop (Humulus lupulus) cultivars in Tasmania, Australia. Virus infections were associated with an increase in the mortality of softwood cuttings following propagation. In all cultivars, height of early growth was a poor indicator of the effect of viruses on cone yield and levels of brewing organic acids (alpha and beta acids). In cv. ‘Nugget’, infection by the virus combinations studied was not associated with reductions in cone yield, however plants infected by PNRSV‐I, in 2000, had 11% lower alpha acids and 7% lower beta acids. In ‘Opal’, infection by HpLV and HpMV were the most deleterious to cone yield, however the effect of HpMV was ameliorated when in combination with PNRSV‐I. Reductions in alpha and beta acid content were attributable only to mixed infections of HpLV + HpMV in combination with either serotype of PNRSV. In ‘Pride of Ringwood’, yield loss was mostly attributable to HpMV and to a lesser extent, HpLV. Some ameliorations in cone yield loss occurred in plants containing a mixed infection between HpMV and HpLV or either of the ilarvirus serotypes. Both of the ilarviruses and HpMV caused reductions in alpha acid content. In ‘Victoria’, cone yield loss was mostly attributable to combinations of viruses such as HpLV + PNRSV‐I and HpLV + HpMV. The deleterious effect of HpLV + HpMV was ameliorated by PNRSV‐A and to a lesser extent, PNRSV‐I. Infection by the virus combinations studied did not significantly affect alpha and beta acid levels in either year. Results suggested the effect of viruses and their combinations differed between cultivars and varied between seasons. This information, when combined with knowledge of the rates of virus re‐infection, can be used to recommend control strategies for the Australian hop industry.     

Studies on rose mosaic disease in field-grown roses produced in the United Kingdom

Arabis mosaic virus (AMV) and prunus necrotic ringspot virus (PNRSV), separately or together, caused in field-grown roses the range of symptoms recognised as rose mosaic disease. PNRSV infection alone generally induced chlorotic line patterns, ring-spots or mottles in the leaves at some time during the growing season; AMV plus PNRSV normally caused chlorotic vein-banding. However, during prolonged periods of high temperatures (c. 21 °C or more) vein banding occurred in some roses infected only with PNRSV. Isolates of PNRSV from rose had particles which were similar in shape, protein mol. wt, density and sedimentation coefficients to previously described isolates of PNRSV from cherry, plum and rose; all were cherry serotypes. In graft-inoculated roses, apple serotypes of PNRSV induced stunting and chlorosis, puckering and distortion of leaves, which closely resembled symptoms associated with rose mosaic in the USA and chlorotic mottle rose mosaic in New Zealand. To avoid possible confusion in using the name rose mosaic it is suggested that the virus(es) present in roses should be named.     

Serological and biological variations of African cassava mosaic virus, in Nigeria

To determine the occurrence of variants of African cassava mosaic virus, 316 cassava leaf samples were collected from mosaic‐affected cassava plants in 254 farmers. fields in 1997 and 1998, covering the humid forest, coastal/derived, southern Guinea and northern Guinea savannas and arid and semi‐arid agroecologies of Nigeria. The samples were tested in triple antibody sandwich enzyme‐linked immunosorbent assay using a panel of 10 monoclonal antibodies (MAbs) against the virus in which 29 reaction patterns were observed. In cluster analysis, nine serotypes were obtained at 0.80 Jaccard similarity coefficient index in which at least 50% of isolates of each serotype reacted alike. The serotypes ranged between two extremes: serotype 1 with 90% isolates reacting with the 10 MAbs and serotype 8 in which 90% of its isolates failed to react with the antibodies. Isolates of serotypes 1, 2, 4 and 8 were widely distributed while those of the other serotypes were estricted to certain agroecologies. Four representative isolates 227 (serotype 1), 231 (serotype 2), 235 and 283 (serotype 8) elicited different responses in Nicotiana, benthamiana, with isolate 283 not able to infect this and other test plants used. The serological variations did not necessarily reflect the biological variations. In polymerase chain reaction tests, one out of the five pairs of ACMV primers tested distinguished only isolate 283. The humid forest, derived/coastal and southern Guinea savannas where most of the crop is grown in Nigeria had a high number of variants, which makes the agroecologies suitable for the selection of resistant cassava clones against ACMV.     

Reproductive Fitness and Random Amplified Polymorphic DNA Variation among Isolates of Pratylenchus vulnus

The reproductive fitness of seven isolates of Pratylenchus vulnus from different geographical areas and hosts was assessed in monoxenic cultures (carrot), and greenhouse cultures (plum, sour orange, and quince). The genetic makeup of the different isolates was compared by Random Amplified Polymorphic DNA (RAPD-PCR). The apple (PvAP-S) and apricot (PvAT-F) isolates reproduced less in monoxenic cultures than the rose (PvRO-S) and walnut (PvWA-A and PvWA-U) isolates. On plum, the rose isolate (PvRO-S) reproduced better than the apple (PvAP-S) and walnut isolate from the United States (PvWA-U). On sour orange, the apple (PvAP-S), unknown origin (PvU-UK), and walnut isolate from Argentina (PvWA-A) multiplied well, whereas the walnut isolate from the United States (PvWA-U), apricot (PvAT-F), and rose (PvRO-S) did not. On quince, the apple (PvAP-S) and walnut (PvWA-U) isolates showed a higher reproduction than the one from unknown origin (PvU-UK). RAPD-PCR patterns among the seven P. vulnus isolates were similar, although high intraspecific varibility was detected. Very few bands of P. neglectus were shared by any population of P. vulnus. A high degree of similarity was found among the patterns corresponding to the rose (PvRO-S), apple (PvAP-S), walnut from the United States (PvWA-U), and unknown origin (PvUK-U) isolates. The apricot isolate (PvAT-F) was the most dissimilar among the seven isolates. No correlation could be established between the genetic variation of P. vulnus detected by RAPD-PCR and reproductive fitness. Results demonstrate high genetic varibility between geographically separated populations of P. vulnus.     

Molecular phylogeny of the psittacid herpesviruses causing Pacheco's disease: correlation of genotype with phenotypic expression

Fragments of 419 bp of the UL16 open reading frame from 73 psittacid herpesviruses (PsHVs) from the United States and Europe were sequenced. All viruses caused Pacheco's disease, and serotypes of the European isolates were known. A phylogenetic tree derived from these sequences demonstrated that the PsHVs that cause Pacheco's disease comprised four major genotypes, with each genotype including between two and four variants. With the exception of two viruses, the serotypes of the virus isolates could be predicted by the genotypes. Genotypes 1 and 4 corresponded to serotype 1 isolates, genotype 2 corresponded to serotype 2 isolates, and genotype 3 corresponded to serotype 3 isolates. The single serotype 4 virus mapped to genotype 4. DNA from a virus with a unique serotype could not be amplified with primers that amplified DNA from all other PsHVs, and its classification remains unknown. Viruses representing all four genotypes were found in both the United States and Europe, and it was therefore predicted that serotypes 1, 2, and 3 were present in the United States. Serotype 4 was represented by a single European isolate that could not be genetically distinguished from serotype 1 viruses; therefore, the presence of serotype 4 in the United States could not be predicted. Viruses of genotype 4 were found to be the most commonly associated with Pacheco's disease in macaws and conures and were least likely to be isolated in chicken embryo fibroblasts in the United States. All four genotypes caused deaths in Amazon parrots, but genotype 4 was associated with Pacheco's disease only in Amazons in Europe. Genotypes 2, 3, and 4, but not 1, were found in African grey parrots. Although parrots from the Pacific distribution represent a relatively small percentage of the total number of birds with Pacheco's disease, all four genotypes were found to cause disease in these species.     

Distribution of the SsuDAT1I restriction-modification system among different serotypes of Streptococcus suis

The SsuDAT1I restriction-modification (R-M) system, which contains two methyltransferases and two restriction endonucleases with recognition sequence 5'-GATC-3', was first found in a field isolate of Streptococcus suis serotype 2. Isoschizomers of the R-M system were found in the same locus between purH and purD in a field isolate of serotype 1/2 and the reference strains of serotypes 3, 7, 23, and 26 among 29 strains of different serotypes examined in this study. The R-M gene sequences in serotypes 1/2, 3, 7, and 23 were very similar to those of SsuDAT1I, whereas those in serotype 26 were less similar. These results indicate intraspecies recombination among them and genetic divergence through their evolution.