A study was made of the plant yellows diseases legume little leaf, tomato big bud, and lucerne witches' broom occurring in Australia.
Thin section electron microscopy revealed large numbers of mycoplasma-like bodies in the phloem tissue of diseased plants, but not in healthy control plants. The bodies were highly pleomorphic; spherical forms ranged in size from approximately 80 to 1000 nm, and filamentous forms were up to 1600 nm in length. They were characterized by bounding "unit" membranes approximately 8 nm thick, and an internal structure consisting of central nuclear areas containing DNA-like fibrils, and peripheral cytoplasmic areas containing ribosome-like particles.
Mycoplasma-like bodies occurred mainly in nature sieve tube elements devoid of cytoplasm, but occasionally also in the apparently normal cytoplasm of cells containing nuclei and other organelles. Those wore possibly phloem parenchyma cells. The lumina of some sieve elements were occluded by mycoplasma-like bodies. The occasional presence of mycoplasma-like bodies in phloem sieve pores suggested movement of the bodies between sieve elements by way of the pores.
Mycoplasma-like bodies were also observed in dodder (Cuscuta australis) grown on little leaf-diseased plants, and in individual specimens of the leafhopper vector (Orosius argentatus) shown to transmit the little leaf agent. In dodder, the bodies occurred in mature sieve tube elements and in phloem cells containing apparently normal cytoplasm. In infective leafhoppers the bodies were found in the salivary glands and filter chamber region of the alimentary canals. In the salivary glands, they were observed in only one of tho three types of acini. A technique for the fixation, embedding, and sectioning of whole leafhoppers for thin section electron microscopy was developed. This avoided leafhopper dissection and allowed the examination of the salivary glands in situ. The technique also had the advantages of being simple and rapid.
Symptoms of little leaf disease were suppressed in the new growth of plants sprayed with 100 mg/ml aqueous Achromycin (tetracycline hydrochloride) or Aureomycin (chlortetracycline hydrochloride). Mycoplasma-like bodies were abundant in the phloem sieve tubes of untreated plants, and in plants which re-developed symptoms 2 to 4 weeks after 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 Orosius argentatus, tho little leaf agent was not recovered from symptomless treated shoots, but was readily recovered from diseased shoots which developed after termination of the Achromycin treatment.
Symptomless shoots detached from tomato plants after a period of Achromycin treatment were either grafted to Datura stramonium plants or established as rooted cuttings. Little leaf symptoms appeared in both groups of plants, showing that the originally symptomless tomato shoots were infected with the little leaf agent. Thin section electron microscopy of the symptomless treated tomato shoots did not reveal typical mycoplasma-like bodies, but a few atypical bodies were observed in phloem sieve tubes.
Attempts to culture mycoplasmas from little leaf-diseased plants on cell-free media were unsuccessful.
The results of thin section electron microscopy, and of chemotherapy of little leaf-diseased plants suggest that the little leaf disease is caused by a mycoplasma-like organism, rather than a virus as previously assumed. The evidence for mycoplasmal aetiology may be summarized as follows:
(i) failure to detect virus-like particles in diseased plants by thin section electron microscopy
(ii) the presence of mycoplasma-like bodies in the phloem tissue of diseased plants, infective dodder, and infective leafhoppers, and the absence of such bodies from control material
(iii) the suppression of symptoms in diseased plants sprayed with tetracycline antibiotics
(iv) the correlation of tetracycline-induced symptom suppression with apparent absence of mycoplasma-like bodies from new symptomless shoots (as indicated by thin section electron microscopy), and the failure of leafhoppers to acquire the little leaf agent from these shoots.
The presence of mycoplasma-like bodies in the phloem tissue of plants affected by tomato big bud and lucerne witches' broom diseases suggests that these diseases are also caused by mycoplasmas.
The little leaf, big bud, and witches' broom agents were distinguished on the basis of differences in symptoms and incubation periods in differential host plants. Two strains ("mild" and "severe") of the witches' broom agent were distinguished by differences in the respective symptoms produced in Nicotiana glutinosa and Datura stramonium. The "mild" strain was not transmitted by Orosius argentatus, and had a significantly longer incubation period in tomato than either the little leaf or big bud agents. The "severe" strain v/as apparently identical to the little leaf agent. Both the little leaf and big bud agents were transmitted by Orosius argentatus, but were distinguished on the basis of differences in symptoms produced in Datura stramonium, and also by the significantly longer incubation period of the little leaf agent in Datura stramonium under controlled conditions.
These results suggest that there are at least three strains of yellows pathogens (presumably mycoplasma-like organisms) in Australia.