Symptomless Shoots Research Articles

The potential of grafting with selected stone fruit varieties for management of almond witches' broom.

A lethal disease of stone fruit trees, characterized by proliferation of axillary shoots and witches’ broom symptoms, has caused severe problems for more than 200,000 almond, peach and nectarine plants in Lebanon since the 1990s. The agent associated with almond witches’ broom (AlmWB) was identified as ‘ Candidatus Phytoplasma phoenicium’, belonging to subgroup 16SrIX-B. Management of the disease has relied on uprooting of affected trees. Since no disease-resistant cultivars have been identified, grafting experiments in the field and in the greenhouse we performed to develop a new option for the integrated management of the disease. AlmWB-affected almond trees were grafted with apricot or plum scions, and their growth was symptomless for over 2 years in the field. Similarly, in greenhouse trials, grafting AlmWB-affected almond scions onto seedlings of plum and apricot resulted in growth of symptomless shoots. One year post-grafting in the greenhouse the phytoplasma was not detected by PCR in almond grafted on Angeleno and Red plum and Early Blush apricot used as rootstocks. The phytoplasma was, however, detected in almond scions grafted on Farclo apricot and Jawhara plum, although their growth was symptomless. Shoots developing from Farclo apricot grafted on AlmWB-affected trees in the field showed severe symptoms 2 months after grafting but recovered 3 months later, and remained symptomless for about 2.5 years. Similarly, in the greenhouse trial, the growth of phytoplasma-infected scions grafted on Early blush apricot developed symptoms 2 months after grafting, did not show symptoms 2 months later, and remained symptomless 1 year later. Quantitative PCR analysis of almond scions grafted on Early blush apricot seedlings confirmed reduction of phytoplasma titre from 44 GU/ng DNA to below detection level.

High-Resolution Melting Analysis: a new molecular approach for the early detection of Diplodia pinea in Austrian pine

The differentiation of Diplodia pinea from closely related species, such as Diplodia scrobiculata and Diplodia seriata, and its detection in plant tissue, represented a critical issue for a long time. Molecular screening tools have recently been developed to address this topic. In this study we applied one of the most sensitive and rapid diagnostic screening method so far developed, called High-Resolution Melting Analysis (HRMA), to detect D. pinea in Austrian pine ( Pinus nigra). HRMA exploits differences in the melting behaviour of PCR products to rapidly identify DNA sequence variants without the need for cumbersome post-PCR methods. We developed a HRMA method to detect specific fungal sequences in the mitochondrial small subunit ribosome gene (mt SSU rDNA). The reliability of this technique was firstly assessed on DNA extracted from pure cultures of D. pinea and closely related species. Amplicon differences were screened by HRMA and the results confirmed by direct DNA sequencing. Subsequently, HRMA was tested on DNA from symptomatic and symptomless pine shoots, and the presence of the fungus was also confirmed by both conventional and molecular quantitative approaches. The HRMA allowed the distinction of D. pinea from closely related species, showing specific melting profiles for the each pathogen. This new molecular technique, here tested in a plant–fungus pathosystem for the first time, was very reliable in both symptomatic and symptomless shoots. HRMA is therefore a highly effective and accurate technique that permits the rapid screening of pathogens in the host.

Sources of primary inoculum ofTranzschelia discolorin Australian almond orchards

Sources of primary inoculum of almond rust (Tranzschelia discolor) were investigated. Retained leaves, fruit not removed during harvest, leaf litter on soil and green or lignified shoots were monitored for viable urediniospores between harvest and the emergence of new leaves. Retained leaves were confirmed as the primary source of overwintering inoculum in almonds with up to 27% of the urediniospores viable at the time of new leaf emergence. Viable urediniospores were also found on symptomless green shoots, retained fruit and lignified shoots. The viability of urediniospores on leaf litter rapidly declined to nil within two months. Inoculation of almond stems and fruit showed that both were capable of producing uredinia, but were less frequently infected compared with leaves. Urediniospores from stem or fruit lesions, symptomless green or lignified shoots, or leaf litter are not considered to play a major role in the epidemiology of almond rust. Inoculation of cherry, peach, nectarine and plum plants with urediniospores collected from almond leaves did not produce disease symptoms indicating that other stone fruit are unlikely to provide an alternative overwintering host for almond rust. Removal of retained leaves in combination with strategic fungicide use may help growers to optimise rust control in almonds.

Managing Diplodia Tip Blight of Landscape Austrian Pines

A long-term survey of 449 Austrian pines growing on the University of Kentucky campus revealed that Diplodia tip blight disease killed 84% of the trees during a 15-year period and that the pines developed increasing levels of disease as they aged. Treatments consisting of the fungicides oxycarboxin, debacarb, or tebuconazole or water were injected into the lower trunk and root flare of individual trees in a block of mature, diseased pines for 4 years. The fungicide treatments did not significantly affect disease levels. The causal fungus could be isolated readily from diseased and symptomless shoots regardless of the treatment. Diplodia pinea was very sensitive to tebuconazole and debacarb in in vitro fungicide tests. Basal drenches with paclobutrazol affected shoot growth but did not significantly reduce tip blight disease levels or tree mortality. Under midsouth United States climate conditions, Diplodia tip blight of Austrian pines is destructive and very difficult to manage.

Detection of ‘Candidatus Phytoplasma pini’ in Pinus sylvestris trees in Poland

AbstractAbnormal shoot branching was observed in Pinus sylvestris trees in Poland. These abnormalities resulted in the formation of dense, ball‐like structures with dwarfed needles. The presence of phytoplasma in the needles of branched and surrounding symptomless shoots was demonstrated using nested‐polymerase chain reaction (PCR) with universal primer pairs that amplified phytoplasma 16S rDNA, as well as using restriction fragment length polymorphic analysis of PCR products. Comparison of nucleotide sequences of DNA samples from three P. sylvestris trees with ball‐like structures revealed that their fragments of 16S rDNA were identical. The nucleotide sequence showed more than 99% similarity with the corresponding fragments of sequence of ‘Candidatus phytoplasma pini’.

Detection of a tomato strain of Parietaria mottle virus (PMoV‐T) by molecular hybridization and RT‐PCR in field samples from north‐eastern Spain

Parietaria mottle virus (PMoV‐T) infecting tomatoes from different commercial fields in Catalonia, Spain was detected by dot‐blot and tissue‐printing hybridization and one‐step reverse transcriptase PCR (RT‐PCR). Specific cDNA was amplified by RT‐PCR from mechanically infected Chenopodium quinoa plants and cloned into a plasmid vector. A digoxigenin‐labelled riboprobe was synthesized for molecular hybridization, and a specific set of primers (PMoV‐1F/PMoV‐1R) were designed for one‐step RT‐PCR assays. Material from different parts of PMoV‐T‐infected tomato plants was used for the analysis. The virus was detected on necrotic apical shoots, fruits with symptoms, and symptomless shoots grown from old necrotic branches, but not on the old necrotic branches themselves. Both molecular hybridization and one‐step RT‐PCR procedures were highly specific, and no cross‐reactions were observed with other related ilarviruses. PMoV‐T was detected successfully by molecular techniques on all infected tomato plants collected from commercial fields, although one‐step RT‐PCR was less robust than molecular hybridization. The observed reliability of tissue‐printing hybridization could greatly facilitate both the large‐scale analysis and epidemiological studies of this important pathogen.

アイリスイエロースポットウイルス（ＩＹＳＶ）によるアルストロメリア条えそ病（新称）とウイルスの分布解析

Alstroemeria plants (Alstroemeria sp. and A. ligtu) with necrotic spots and streaks on leaves were found in Chiba prefecture, Japan. Tospovirus-like enveloped, spherical particles 80-100nm in diameter and filamentous particles 750-850nm in length were observed together in the infected leaves. The enveloped particles were identified as Iris yellow spot virus (IYSV) by RT-PCR. Deduced amino acid sequences of the nucleocapsid gene of the two IYSV isolates (designated CbAlsD1 and Als1) were compared with those of IYSV isolates reported previously; the isolates were genetically similar to an isolate from Dutch iris (Iris hollandica) in the Netherlands. A local lesion isolate of CbAlsD1 reproduced the original symptoms on alstroemeria seedlings without the presence of filamentous virions. These results showed that IYSV was the causal virus of necrotic spots and streaks. Naturally infected A. ligtu plants produced new shoots that appeared healthy when grown under thrips-free conditions. Although IYSV could not be detected in these symptomless shoots by RT-PCR alone, the virus was detected by Southern blotting of the RT-PCR products. Therefore, a low level of IYSV virions exist in the symptomless shoots that developed from infected alstroemeria plants.

Detection of Latent Sphaeropsis sapinea Infections in Austrian Pine Tissues Using Nested-Polymerase Chain Reaction

ABSTRACT Sphaeropsis sapinea is the causal agent of Sphaeropsis tip blight disease of pines. Past surveys of diseased and symptomless Austrian and Scots pines revealed that latent infections of symptomless shoots by S. sapinea are common. The role of these latent infections in the tip blight disease is unknown. A sampling technique and nested-polymerase chain reaction (PCR) protocol were developed to detect latent S. sapinea in symptomless pine shoots. The sampling protocol was designed to be minimally destructive to the shoot so it could be preserved for further studies. The primers that were developed were specific for S. sapinea DNA and did not amplify DNA from any of 13 other endophytic fungal species that were commonly isolated from symptomless pine shoots. The PCR primers also amplified DNA of Botryosphaeria obtusa, which was, however, rare in symptomless Austrian pine tissues. The protocol detected as little as 0.93 pg of S. sapinea DNA in terminal bud samples and 10.4 pg of DNA in bark samples. Correlation (chi-square) analyses indicated that the nested-PCR protocol detected latent S. sapinea infections in both bud and bark samples with an efficiency that was statistically equivalent to isolating the fungus from the tissue. The nested-PCR protocol will make it possible to more quickly identify latent S. sapinea infections in symptomless pine shoots and should be useful in future studies of the latency phenomenon.

ONE YEAR SURVIVAL OF ERWINIA AMYLOVORA IN SYMPTOMLESS PEAR SCIONS

Pear scions cv. ‘Abate Fetel’ on B29 rootstock were raised individually in pots from dormant buds. In autumn 1999, the main stems were divided into 5 groups and inoculated at 10 day intervals starting from 21 September with a virulent RifR mutant of Erwinia amylovora. For the inoculation, 30,000 cells of E. amylovora were deposited on a transverse cut 10 cm from the tip. After ensuring establishment of the pathogen, the five groups of plants were placed in the open and inspected weekly for the presence of fire blight symptoms. Scions treated with water were used as a control. All symptomatic scions were eliminated by mid-April 2000. The frequency of scions developing symptoms (21.3%) decreased from the first to the last inoculation date (from 5/15 to 1/15). In autumn, a year after inoculation, the plants remaining symptomless were monitored for the presence of endophytic E. amylovora. Re-isolation on selective medium was successful in 13 plants out of 41. Reisolation was also successful in 7 segments taken from the side shoots developed from the first and second nodes below the inoculation point. The results show that E. amylovora can survive a year in symptomless planted scions. Isolation of the mutant from symptomless shoots developed during the growing season following inoculation indicates that E. amylovora may have a prolonged symptomless endophytic phase.

Latent Infection of Austrian and Scots Pine Tissues by Sphaeropsis sapinea.

This study documents latent infection of Austrian and Scots pine tissues by Sphaeropsis sapinea, the causal agent of Sphaeropsis tip blight disease. Symptomless shoots from diseased or apparently healthy Austrian and Scots pine trees were cultured and analyzed for the presence of S. sapinea. Nearly half of the sampled Austrian and Scots pine trees that appeared to be completely healthy had detectable latent S. sapinea infections. More than half of the symptomless shoots on visibly diseased Austrian and Scots pine trees were also latently infected with S. sapinea. S. sapinea was isolated from symptomless shoot stems, needles, buds, immature cones, and male flowers. In symptomless infected shoots, the fungus was primarily associated with the bark and phloem tissues. Six isolates of S. sapinea from symptomless Austrian and Scots pine shoot tissues were pathogenic on Austrian pine seedlings in the greenhouse. The presence of S. sapinea in symptomless tissues of tip blight-diseased trees has important implications for disease management.

ASSOCIATION BETWEEN A NATIVE SPITTLEBUG (HOMOPTERA: CERCOPIDAE) ON MONTEREY PINE AND AN INTRODUCED TREE PATHOGEN WHICH CAUSES PITCH CANKER DISEASE

AbstractJuvenile Monterey pines, Pinus radiata D. Don (Pinaceae), in a native stand on the Monterey Peninsula, California, exhibited wilted green shoots in the spring of 1996. The pitch canker fungus, Fusarium subglutinans (Wollenweb. & Reinking) P.E. Nelson, Toussoun & Marasas f.sp. pini, was subsequently isolated from 95% of these shoots. Spittle masses produced by Aphrophora canadensis Walley were observed on the symptomless shoots of many of these trees. The pitch canker fungus was isolated from the feeding sites of this insect on 55% of symptomless shoots, and from shoot sections adjacent to these feeding sites on 29% of the shoots. Shoots with spittlebugs feeding on them in May 1996 were more likely to develop pitch canker disease by September 1996 and March 1997 than shoots without spittlebugs. Shoots with spittle masses in March 1997 were as likely to develop pitch canker disease by May and August 1997 as shoots without spittle masses, but the origin of the infection was most likely where A. canadensis feeding had taken place. In a controlled test, the incidence of pitch canker on shoots of potted Monterey pines was dependent on the presence of a spittlebug and a spore suspension of the pathogen. Thus, both field observations and controlled studies show an association between native A. canadensis and the introduced pitch canker pathogen. The role of A. canadensis in the epidemiology of pitch canker disease remains to be determined.

Factors determining recovery and reversion in mosaic‐affected African cassava mosaic virus resistant cassava

SummaryThe severity and persistence of symptoms of mosaic virus disease were monitored during the first six months of two growing seasons in cassava of the African cassava mosaic virus (ACMV)‐resistant cv. TMS 30572 either inoculated by grafting with a mild or severe strain or infected from the planted cutting. Symptomless shoots developed between January and March 1995 in two field trials differing in age by c. 6 months; this recovery occurred during particularly hot weather. Recovery was often only temporary in the plants inoculated with the severe strain and occurred later compared with those inoculated with the mild. In 1996, the weather was cooler and recovery that year was delayed until flowering, c. 7 months after planting, when recovered shoots were often produced from buds in the axils of symptomless leaves produced amongst diseased leaves. Most cuttings taken from the upper parts of diseased plants produced symptomless (reverted) progenies whereas most cuttings taken from the base of diseased plants produced diseased progenies. Reversion seemed to be associated with the recovery that had already occurred in the upper stems of the parent plants.

Subterranean Clover Distortion: A New Viruslike Disease with Unusual Characteristics

A new mechanically transmissible viruslike disease, subterranean clover distortion, was found in subterranean clover (Trifolium subterraneum) in a pasture in New South Wales. The pathogen has a wide experimental host range. Symptoms varied from thickening and distortion of young leaves, reddening of older leaves, and production of axillary shoots on subterranean clover to chlorosis of leaves, flower malformation, flower abortion, and stem necrosis on other hosts. Symptomless shoots often developed on infected plants. No local lesion host was identified (.)

Recovery Resistance to Downy Mildew in Pearl Millet

The development of symptomless shoots from pearl millet plants that show typical systemic symptoms of downy mildew (DM) disease is reported. This phenomenon, referred to here as “recovery,” and the trait as “recovery resistance,” was detected in 28 of the 33 pearl millet genotypes studied. These represent a wide range of breeding materials and cultivars. The genotypes showed considerable variability for the frequency of plants with recovery resistance and for the extent of sexual and asexual sporulation of Sclerospora graminicola in recovered plants before recovery. The levels of recovery resistance were substantially increased in several genotypes through pedigree selection under high disease pressure in the DM nursery. Injection of growing points of the symptomless shoots of recovered plants with a sporangial suspension, or clipping the stems and shoots of recovered plants growing under high disease pressure, generally did not result in subsequent disease development. Lines produced from recovered plants maintained their resistance at three locations in India. The phenomenon was also observed in Mali and Niger. It is suggested that recovery resistance may be an effective defense mechanism that should be exploited in breeding pearl millet for durable resistance to DM.

Virus-Free Shoots from Cassava Stem Cuttings Infected with Cassava Latent Virus

whiteflies were killed with malathion Pacumbaba, R. P. 1985. Virus-free shoots from cassava stem cuttings infected with cassava latent sprays after 8 hr of feeding on the test virus. Plant Disease 69:231-232. plants. Ten and 15 symptomless, unsevered Cassava stem cuttings infected with cassava latent virus (CLV) gave rise to shoots that showed CLV shoots in the first and second year of the symptoms at emergence, shoots that showed CLV symptoms 3-5 wk later, and shoots that remained symptomless after 6 wk of observation. CLV symptoms appeared 5-11 days earlier when experiment, respectively, were also symptomless shoots were detopped than when they were left intact. Symptomless shoots severed 2 detopped the third week after shoots wk after emergence from CLV-infected stem cuttings and subsequently detopped did not develop emerged from the ground. An equal CLV symptoms. These plants, however, readily developed symptoms of CLV when colonized with number of unsevered symptomless shoots CLV-laden Bemisia tabaci. Therefore, symptomless shoots were considered free of CLV and could that were not detopped served as be used as healthy planting stock. controls.

Effects of Tetracycline Antibiotics on Plants Affected by Legume Little Leaf Disease

Nicotiana glutino8a, aster, and tomato plants affected by legume little leaf disease were treated by spraying the foliage with 100 ,..,g/ml aqueous solutions of either Achromycin (tetracycline hydrochloride) or Aureomycin (chlortetracycline hydro-chloride), every 2-3 days for periods of 4-8 weeks. Disease symptoms were sup� pressed in the new growth of all plants during the treatment period, but reappeared 2-4 weeks after the last application of antibiotic. Mycoplasma-like bodies were abundant in the phloem sieve tubes of untreated aster, and in aster which redeveloped symptoms after the antibiotic treatment had been discontinued. In contrast, Mycoplasma�like bodies could not be detected by thin� section electron microscopy in symptomless shoots of Achromycin-treated plants immediately after a period of treatment. In concomitant leafhopper transmission tests with the vector Orosius argentatus, the little leaf agent was not recovered from the symptomless treated shoots, but was readily recovered from diseased shoots which developed after termina-tion of the Achromycin treatment.

VIRUS DISEASES OF THE POTATO: THE “CARRIER” PROBLEM. ITS RELATION TO SYMPTOMATOLOGY AND COMMERCIAL POTATO GROWING

SUMMARY Symptomatology, of which a study of “carriers” is regarded as one aspect, tends to be used as a means of segregating “unit” viruses rather than as a means of furthering plant physiological studies. The effects of changes in environmental conditions of growth, acting through the unstable metabolism of a plant upon a virus, have not been adequately studied. Until this is done, the specificity of the reactions said to be due to a unit virus must remain in doubt and, with it, the whole foundation of the biological methods of segregating viruses. The symptomatological unit is the plant cell, and the clinical picture resulting from infection is the expression of the varying degree to which invaded cells dominate, or are dominated by, a virus. Successive stages in plant dominance are shown by (a) discontinuous or local symptoms; (b) symptomless shoots; (c) individual carrier plants in a variety usually reacting to infection; (d) carrier varieties. The conditions under which the power to suppress symptoms can be broken down are discussed. Masked symptoms are revealed by relatively slight environmental changes, but the breakdown may be the more difficult to achieve as perfect “carrying” is approached. There is no fundamental difference between “masking” and “carrying”. The conditions under which masking can be converted into carrying are also discussed. Evidence is submitted to show that a perfect carrier plant is not only unaffected in vigour as a consequence of carrying a virus, but may thereby exhibit a reduced susceptibility to certain other viruses in the field. Field trials are described in which proof is given that the viruses carried by the varieties King Edward VII (paracrinkle), and Up‐to‐Date (streak and virus X), were not transmitted to intolerant varieties under the most favourable conditions. These carrier varieties are not therefore the serious menace to other varieties they are commonly believed to be.