Mycoplasma-like organisms inLupinus hybrids showing witches’ broom symptoms

A previously undescribed disease onLupinus hybrids manifesting typical witches broom symptoms occurred spontaneously in a locality in North Bohemia. Broomed shoots appeared only in late summer and resulted from the abnormal proliferation of dormant axillary buds in leaf and fruit axils. The shoots were bushy and their leaves were dark green and not reduced in size. Electron micrographs of ultrathin sections showed the presence of numerous mycoplasma-like organisms (MLO) in diseased but not healthy phloem tissues.     

Mycoplasma-like Organisms in Narcissus sp.

Abstract The use of electron and optical fluorescent microscope confirmed the presence of mycoplasma-like organisms (MLO) in Narcissus sp. plants showing leaf chlorosis, phyllody and degenerated flowers. Unidentified multivesicular membrane bounded bodies were also found in some sieve tubes of the diseased plants.     

Occurrence of Anomalous Mycoplasma-like Organisms in Grapevine Yellows-diseased Phloem

Pathological changes in Vitis vinifera cv. Caveccia phloem from leaves showing symptoms of a flavescence doreé-like disease consisted of obliteration, necrosis and collapse of the sieve elements and associated companion cells, and excessive callose accumulation in lateral sieve areas and sieve plates of apparently normal mature sieve elements. Unusual structures, also found in degenerate sieve elements of diseased leaf vein specimens, were strongly electron-dense and bounded by a unit membrane or an electron-transparent border, and considered to be senescent forms of mycoplasma-like organisms. The significance of these findings in relation to possible host responses to the yellows pathogen is discussed.     

Occurrence of Anomalous Mycoplasma-like Organisms in Grapevine Yellows-diseased Phloem

Pathological changes in Vitis vinifera cv. Caveccia phloem from leaves showing symptoms of a flavescence doreé-like disease consisted of obliteration, necrosis and collapse of the sieve elements and associated companion cells, and excessive callose accumulation in lateral sieve areas and sieve plates of apparently normal mature sieve elements. Unusual structures, also found in degenerate sieve elements of diseased leaf vein specimens, were strongly electron-dense and bounded by a unit membrane or an electron-transparent border, and considered to be senescent forms of mycoplasma-like organisms. The significance of these findings in relation to possible host responses to the yellows pathogen is discussed.     

Mycoplasma-Like Organisms in Clove Trees Affected by Sudden Death Disease in Zanzibar and Pemba

Mycoplasma-like organisms were seen in eight of twenty trees affected by sudden death disease from the islands of Zanzibar and Pemba. Vesicular bodies were also seen in the phloem of a further six affected trees but did not possess all the characteristics of mycoplasma-like organisms. No virus-like particles were found in any of the twenty sudden death trees examined.     

Mycoplasma-like Organisms Associated with Little Leaf Disease of Eucalyptus microtheca Muell.

A little leaf disease has been observed in several plants of Eucalyptus microtheca Muell. grown in Wad Medani area, Sudan. The disease is characterized by general plant stunting, reduction in diameter and height of trunks of affected trees, yellowing and reduction in leaf size, shortening of internodes together with excessive proliferation of axillary shoots. Electron microscope observations on ultrathin sections have revealed the presence of pleomorphic bodies in the sieve elements of diseased but not of healthy plants. These bodies possess DNA strands, ribosome-like granules and a three-layered unit membrane well defined at high magnifications (> 100,000) suggesting a mollicute nature of these bodies. Studies using semi-thin sections revealed the absence of helical forms. Thus, a mycoplasma-like organism (MLO) is suspected to be the agent responsible for the disease. The results are discussed with reference to the economic and environmental importance of this tree species in the Sudanand, the possible threat of the disease to its cultivation.     

桑萎缩病的类菌原体病原物及其超微病变特征

对感染桑萎缩病的桑叶及嫩梢进行超薄切片的电镜观察发现,其韧皮部组织,筛管及伴胞内有多型性类菌原体。菌体为圆形及椭圆形,大小约为50～160nm,双层膜,厚度约为8～10nm,内含物中具有核质样的纤维状物质,而在健株叶梢中未观察到任何病原体．随着病害的发展,可观察到细胞成份的降解。第一,在感病植株叶肉细胞内存在部分细胞核的降解、核膜破裂或核质流失甚至核仁分散消失。第二,叶绿体内有不同程度的淀粉粒和嗜锇颗粒累积,部分叶绿体外膜破裂,基质流失,基粒降解。第三,线粒体及粗面内质网在数量上有所增加,部分线粒体嵴已降解。     

Cytopathology of Plants Infected with Viruses. Ultrastructure of the Leaf Cells of Cereals Affected by Cereal Rhabdoviruses

Ultrathin sections of oat, wheat, and ryegrass leaves from healthy plants and plants infected with rhabdoviruses by leafhoppers Laodelphax striatellusFallen were studied under the electron microscope. The bacilliform virions often surrounded by endoplasmic reticulum (ER) membranes, viroplasm, and tubular structures conforming, in diameter and structure, to the rhabdovirion nucleocapsid were observed in the cytoplasm of leaf cells of the diseased plants. The cereal pseudorosette virus [(165–200) × (63–70) nm, CPV] is the causative agent of the disease of cereals in Siberia. The mycoplasma-like organisms were found in the phloem cells of plants infected with CPV. The cereal mosaic virus [(360–420) × (56–64) nm, CMV] is the causative agent of the disease of cereals in the Russian Far East. CMV appears to be a strain of the northern cereal mosaic virus.     

Alfalfa witches’ broom in Czechoslovakia and demonstration of mycoplasma-like organisms in the affected plants

Alfalfa witches’ broom manifested by an expressive increase in the number of stems and dwarfing of the affected plants was found in Czechoslovakia. The disease is not saptransmissible and was transmitted by grafting to healthy alfalfa plants. The disease occurrence is sporadic. The symptoms observed in comparison with those described in Australia and USA are mild and inconspicuous. Electron micrographs of ultrathin sections showed the presence of numerous mycoplasma-like organisms (MLO) in diseased but not in healthy phloem tissues.     

Mycoplasma-Like organisms associated with stunting ofGypsophila paniculata L.

A new undescribed yellows-type disease ofGypsophila paniculata L. was found. Since a stunted growth is the predominant symptom the disease has been termed stunting. Electron micrographs of ultrathin sections revealed numerous mycoplasma-like organisms in diseased phloem tissues. Attempts to maintain the diseasedGypsophila plants for further investigation of this disease failed for the present in spite of all applied modifications in nutrient media.     

Banded filaments associated with the aster yellows MLO in Vinca rosea L

The ultrastructure of an aster yellows mycoplasma-like organism was studied in the phloem of periwinkle (Vinca rosea L.) plants. Banded filaments were observed in association with mycoplasma-like organisms of characteristic morphology. The filaments were variable in length, from 50-100 nm in width, and displayed a regular periodic banding of alternating electron-dense and electron-lucent structures.     

Antholysis in Cassava (Manihot esculenta Crantz) Possibly Caused by Mycoplasma-Like Organisms

Phyllody and apostasis of cassava plants were frequently observed during recent disease surveys in the Cauca Valley of Colombia. Many valuable cassava clones have been affected rendering them unsuitable for hybridization. Light and electron microscopic observations have revealed the presence of mycoplasma-like organisms in the diseased phloem tissues. The causal agent is sensitive to tetracycline and streptomycin at 1000 ppm a. i., but not to penicillin.     

罗汉果疱叶丛枝病的病原及其在某些寄主上的症状反应

葫芦科的罗汉果(Siraitia grosvenori(Swingle)Jeffrey)感染一种系统性的黄化型病害。 通过两次超薄切片的电镜观察:在病茎和病叶脉的韧皮组织中的薄壁细胞和伴胞中,发现有类菌质体(MLO),同时在重复的电镜观察中,在上述组织的薄壁细胞内,还发现风轮状的病毒(Virus)内含体。在健株的上述组织中。没有发现MLO和风轮状内含体。 在用三种不同接种方法(媒介昆虫、叶擦和汁液重创摩擦)进行的生物学测定的结果表明:罗汉果疱叶丛枝病有广泛的寄生范围,其症状特点:在常用的鉴定病毒的常规寄主上,最终均呈现黄化症状,没有系统性或局部性的斑点反应。其中在葫芦科的某些植物上的症状,基本上与罗汉果的症状相近似:初期明脉或脉间褪绿,继之出现疱状斑驳叶,叶片扭卷畸形,后期黄化。在茄科的供试植物上,症状特点:初期多为脉间褪绿,随之株形矮化,叶片变小。豆科上的症状:既有明脉,也有脉间褪绿,叶片呈现细长扭曲,变小并丛生。十字花科的症状特点是褪绿斑驳叶,株形矮化,叶片变小。     

The aster yellows controversy: current status

Evidence for and against the spiroplasmal etiology of aster yellows (AY) disease is examined. A spiroplasma, serologically identical to Spiroplasma citri, was cultivated by some workers from lettuce (Lactuca sativa L.) plants claimed to be naturally infected with AY. The isolated spiroplasma was shown to be infectious by injecting Macrosteles fascifrons with the cultured organisms and then confining the injected leafhoppers on healthy plants. The reports claiming that a spiroplasma is the etiological agent of AY, however, exist only in astract form, and several essential questions still need to be answered to substantiate the claim. Evidence against the claim is based on significant differences that have been observed between the behavior of S. citri and the AY agent in the leafhoppers as well as in the plant. Also, helical organisms could not be found in AY-infected plants by either scanning or immunosorbent electron microscopy, and S. citri is serologically unrelated to the mycoplasma-like organisms found in AY-infected plants. These results strongly support the conclusion that the classical AY disease is not caused by a variant of S. citri.     

Multiplication and morphology of Spiroplasma citri in the leafhopper Euscelis plebejus

Spiroplasma citri multiplied in all Euscelis plebejus leaf hoppers injected and sometimes reached titres of over 1 × 10⁷ colony forming units per insect. Spiroplasmas could be isolated from the haemolymph at all times although helices were only apparent for a few days after injection. The salivary glands of injected insects contained membrane bound pockets densely packed with mycoplasma-like bodies. These bodies were frequently infected with virus-like particles similar to those found in cultures of S. citri. Spiroplasmas had little effect on the longevity of the leafhoppers.     

Gladiolus “grassy top” disease recorded in Czechoslovakia and increased susceptibility of affected corms toFusarium sp.

Gladiolus “grassy top” disease was recorded in Czechoslovakia. Electron micrographs of ultrathin sections prepared from dwarfed leaves sprouting in spring from corms of affected gladiolus plant showed a very high accumulation of mycoplasma-like organisms (MLO) in sieve tubes. These corms were stored during the winter period in a cold glasshouse and their overground plant parts were removed only before planting in spring. In autumn, when the corms were dug up in the field, those of affected plants were mostly infected withFusarium sp. while those of apparently healthy ones were intact. This observation may indicate a conspicuously increased susceptibility of affected corms to the fungus.     

Relation of in vivo morphology to isolation of plant spiroplasmas

Two procedures were developed to isolate plant spiroplasmas directly onto DG-2 agar plates or in DG-2 broth without subcultures or dilutions. The frequency of successful spiroplasma isolations was increased by centrifuging samples, after passing through a 0.45-μm filter, at 25,000 × g for 1 h. Spiroplasmas were obtained from peach, cherry, Madagascar periwinkle, and celery with typical symptoms of the Green Valley strain of X disease (GVX), from peach with typical symptoms of the peach yellow leaf roll strain of X disease (PYLR), from Madagascar periwinkle with typical symptoms of aster yellows (AY), from celery with atypical symptoms of GVX (mild GVX), from plantago with atypical symptoms of aster yellows (mild AY), and from stubborn-diseased citrus. Isolations were consistent (>90%) from plants with mild GVX, mild AY, and citrus stubborn, while isolations were inconsistent (0–9%) from plants with typical symptoms of GVX, PYLR, and AY. The role of the isolated spiroplasmas in plant disease was not determined in this study. All spiroplasma isolates were serologically indistinguishable fromSpiroplasma citri. Spiroplasmas were seen in electron micrographs of 8 out of 9 examined plants from which spiroplasmas were isolated. However, electron micrographs of all 13 examined plants from which no spiroplasmas were isolated contained mycoplasma-like organisms (MLOs) but no, spiroplasmas. These results indicate that there is a correlation between helical MLOs in vivo and successful isolation of spiroplasmas, and that plants may be infected with bothS. citri and nonhelical mycoplasmas.     

Isolation, Chemical Composition, and Ultrastructural Features of the Cell Membrane of the Mycoplasma-Like Organism Spiroplasma citri

Thin sections of Spiroplasma citri, a mycoplasma-like organism isolated from citrus infected with "Stubborn" disease, showed the organisms to be limited by a single trilaminar plasma membrane. An additional outer layer could, however, be frequently seen in freeze-etched preparations of unwashed cells. The organisms were found to be extremely sensitive to lysis by osmotic shock. The cell membrane of S. citri isolated in this way resembled that of mycoplasmas in ultrastructure and gross chemical composition. The isolated membranes showed the characteristic trilaminar shape in section and the typical particle-studded fracture faces in freeze-etched preparations. Protein and lipid formed over 80% of the total dry weight of the membrane, which had a density of ~1.180 g/cm(3). Cholesterol constituted over 20% of the total membrane lipid. Phosphatidyl-glycerol, synthesized by the organisms, was the major phospholipid. Significant amounts of hexosamine (15 to 35 mug/mg of membrane protein) could be found in the membrane preparations. Our results support the thesis that S. citri does not possess a cell wall, either of the gram-positive or the gram-negative type, though it may be coated by some other type of an envelope or by a slime layer, at least temporarily.     

Isolation and characterization of full-length chromosomes from non-culturable plant-pathogenic Mycoplasma-like organisms

We describe the isolation and characterization of full-length chromosomes from non-culturable plant-pathogenic, mycoplasma-like organisms (MLOs). MLO chromosomes are circular and their sizes (640 to 1185kbp) are heterogeneous. Divergence in the range of chromosome sizes is apparent between MLOs in the two major MLO disease groups, and chromosome size polymorphism occurs among some related agents. MLO chromosome sizes overlap those of culturable mycoplasmas; consequently, small genome size alone cannot explain MLO non-culturability. Hybridization with cloned MLO-specific chromosomal and 16S rRNA probes detected two separate chromosomes in some MLO ‘type’ strains. Large DNA molecules that appear to be MLO megaplasmids were also demonstrated. The ability to characterize full-length chromosomes from virtually any non-culturable prokaryote should greatly facilitate the molecular and genetic analysis of these difficult bacteria.     

Rapid preparation of DNA from phytopathogenic mycoplasma-like organisms for polymerase chain reaction analysis

A simple procedure for the processing of MLO (mycoplasma-like organisms)-infected plant samples for PCR analysis is described. It is based on differential centrifugation and removal of enzyme inhibiting agents with the aid of a resin. Results of the amplifications are comparable with those achieved with more complex DNA extraction procedures. The method does not require any extraction with organic solvents or alcoholic precipitation of nucleic acids.