GREENING, STUBBORN AND RELATED DISEASES Penicillin and Tetracycline Treatments of Greening Disease-affected Citrus Plants in the Glasshouse, and the Bacterial Nature of the Procaryote Associated with Greening J. M. Bod, P. Bonnet, M. Garnier, and B. Aubert DEDICA TED TO RALPH E. SCHWARZ (1929 - 1979) Since the discovery of the procaryote brane is a cell wall. associated with citrus greening disease Recently, Gibbons and Murray (1978) (Lafleche and BovC, 1970a and b), our have proposed that the kingdom Pro- group has established the following caryotae be divided into four divisions facts. 1) Under normal electron micro- according to the presence of peptidogly- scopy, the envelope surrounding the can (PG) and the type of cell wall. PG is organism is comprised of three zones: a the specific constituent of most bacterial dark, electron-absorbing inner zone, a cell walls. The division Gracilicutes is dark outer zone, and an intermediate, comprised of organisms having a Gram- clear, electron transparent zone. The negative, membranous cell wall contain- thickness of the three zones is approxi- ing PG. The Firmacutes include the mately 250 A. Such an envelope is far prokaryotes with a Gram-positive, PG- too thick to be a single unit membrane, containing, nonmembranous cell wall. and the mycoplasma nature of the green- The Mendocutes have a cell wall, but no ing organisms was questioned (Saglio et PG; halophilic bacteria belong to this al., 197 1; Bove and Saglio, 1974). 2) The division. The Mollicutes have no cell various geographical forms of greening wall or PG; they are surrounded only by (blotchy mottle, citrus decline, leaf a cytoplasmic membrane, shared by all mottling, yellow branch, and likubin), living cells. are all characterized by the same 250 The greening organism (GO) has a A-thick envelope system (Bove and cell wall, and is clearly not a mollicute. Saglio, 1974; Garnier et al., 1976). 3) With its membranous cell wall, the GO While the inner and outer dark, electron- is obviously not a firmacute, but could dense zones are often parallel, they are belong to the Gracilicutes or to the clearly separated, suggesting that each Mendocutes. The typical triple-layered is a single membrane (Garnier et al., outer membane of the GO suggests its 1976). 4) Each of the two electron-dense inclusion in the division Gracilicutes. If zones can be resolved into a >riple- a gracilicute, the GO must contain PG. layered unit membrane, 90-100 A thick Hence, the presence or absence of PG (Garnier and BovC, 1977), confirming would determine the classification of an earlier report by Moll and Martin the GO. The PG of gracilicutes is gener- (1974). The inner membrane is the cyto- ally seen as an electron-dense zone, plasmic membrane, the outer mem- sometimes called R layer, located be- Eizhrh IOCV Conference tween the cytoplasmic membrane and solution (final pH: 7.2) devised for the membranous cell wall. Such a zone citrus by P. Smith (USDA, Orlando, could not be observed in the GO (Moll Florida). The plants were kept in a glass- and Martin, 1974; Tanaka and Doi, house between 20 and 27OC, and illu- 1976). However, we have occasionally minated for 16 hours with supplemental noticed that, at certain locations, the fluorescent light in winter. They were inner layer of the outer membrane is watered with nutrient solution (with or somewhat thicker, more electron-dense, without antibiotics) once every 1 or 2 than the other layers, reminiscent of the weeks, as needed. Sodium benzyl- PG zone of certain gracilicutes (Garnier penicillin (Penicillin G) was from Specia, and Bovt, 1977). Since the GO is not Paris, France and tetracycline-HCl available in culture, direct biochemical from Pfizer, Amboise, France. The detection of PG has not been achieved; presence of penicillin or tetracycline in however, indirect indications for the leaves of the treated plants was checked occurrence of -PG in the GO can be ob- according to Aubert and Bovt (1980) tained by studying the effect of penicillin after each immersion of the roots into on greening-affected citrus plants. This the antibiotic solution. The concentra- antibiotic inhibits a late step (transpep- tion of penicillin in the leaves was ap- tidation) in the biosynthesis of PG proximately one-hundredth (1 / 100) (Ghuysen and Shockman, 1973). that of the solution used for root immer- In this paper, we report that greening- sion. Electron microscopy techniques affected sweet orange plants outgrow have been described earlier (Garnier symptoms when treated with penicillin and Bovt, 1977). G and that no GO's can be seen in the RESULTS sieve tubes of new, symptomless leaves. However, the GO's and the symptoms Experiment I. The following plant reappear when the treatment is stopped. material was used: From these results, we conclude that the a. Healthy: two Hamlin and four greening organism probably possesses Madam Vinous sweet orange seedlings. PG and belongs to the division Gracili- b. Greening-affected: six Hamlin cutes. Preliminary reports of this work seedlings infected in 197 1-1 973 with have already appeared (BovC, 1978; likubin (PD-TY-lo), six Hamlin seed- Garnier and BovC, 1978; Bovt, 1979). lings infected in 1970-1974 with Indian greening (Poona), two Hamlin seedlings MATERIALS AND METHODS infected in 1970 with leaf mottling (Lipa In the first experiment, we used G 3), two Hamlin seedlings infected in diseased citrus seedlings which had been 1974 with Reunion greening, and four infected with the greening pathogen for Madam Vinous seedlings infected in many years. Seedlings infected with 1970 with South African greening (G I Spiroplasma citri and the leaf curl A S). pathogen were compared to greening- c. Leaf curl-affected: four Madam affected plants. The second experiment Vinous seedlings infected in 1972 with was carried out with healthy or leaf curl (Campinas), two Orlando greening-affected sweet orange buds tangelo seedlings infected in 1975 with propagated on healthy rootstocks. The leaf curl (Campinas). effect of tetracycline-HC1 as well as d. Stubborn affected: four Madam penicillin was investigated. Healthy and Vinous seedlings infected in 1970 with greening-infected Madam Vinous sweet C159 stubborn (courtesy Prof. E. C. orange seedlings were used in the third Calavan). experiment. Half of each group of seedlings was All plants were grown in sterilized treated with penicillin, and the other white sand in 5 (expt. 111)- or 10 (expt. I half left untreated as controls. The and 11)-liter plastic containers. Penicillin outline of the antibiotic treatment in or tetracycline was applied in a nutrient expt. I is given in fig. 1 (expt. I). Greening, Stubborn and Related Disea.se.s EXPT. I TIME t 7 1 b t t T6 .7 H 1 5 1 MONTH EXPT. I1 M (SHOOTS) M (SHOOTS) 8/3/77 M (SHOOTS) P (ROOTS) P (PLANTS) H T5 6 T7 T8 1 MONTH 3/20/78 8/17/78 4/24/79 EXPT. 111 GRAFT INOCULATION M M TIME * t H 1 MONTH Fig. 1. Outline of antibiotic treatments - Expt. I. At TI,Ti and TTthe roots of sweet orange seedlings were immersed for 17 hr in a nutrient solution containing penicillin-(; (I gfliter). From TIto Tz the seedlings were watered with nutrient solution containing penicillin-(; (0.2 gfliter). At T4 (June 6,1977) and Th (July 25,1977) the number of new leaves and the length of new shoots were measured (M); at T7 (August 17,1977) the root system was photographed (P). Expt. 11. Buds were propagated August 3,1977 on seedlings in pots. At TI,Tz, T?,T4, Th the pots were immersed in nutrient solution containing penicillin-(; (1 gfliter) or tetracycline-HCl (1 gfliter) for 17 hr. From Th to T7 plants were watered with nutrient solution containing penicillin-<; (0.2 gfliter) or tetracycline-HCl (0.05 gfliter). Expt. 111. From TI to Ti plants were watered with nurtient solution containing penicillin-(; (0.5 gfliter). At Tz (October 20,1978) and Tq (January 25, 1979) photographs were taken (P). At T4 and Tq (March 27, 1979) length of shoots was measured (M). Control plants in all experiments were treated with nurtient solution only. Ei~hthIOCV Conference The number of new leaves and the shoots had grown as well as those of the length of new shoots were measured 11 penicillin-treated plants, and far more weeks (T4) and 18 weeks (T6) after the than those of the untreated controls beginning of the experiment (TI). There (table 2; fig. 3, GT compared to GS and was no significant effect of penicillin on GP). The development of healthy buds the healthy plants, but the treated was first inhibited by penicillin, but greening-affected seedlings, all types, later they grew as well as the untreated grew shoots averaging twice as long as controls (table 2; fig. 3-H, P compared those of untreated plants, and produced to S). Growth of the greening-affected many more leaves (table 1). The penicil- buds was greatly favored by penicillin lin treatment (+ P) considerably im- treatment (table 2; fig. 3, GP compared proved the root system of the greening- to GS). On greening-affected plants, affected plants (Gl, G2, G3) after 21 penicillin was as effective as tetracycline weeks but had no influence on the well- on a number of plants, but its effect was developed root system of the healthy less uniform (fig. 3, GP compared to plants (H) (fig. 2). GT). No leaf symptoms of greening were Penicillin had no effect on stubborn- observed on the antiobiotic-treated or leaf curl-affected seedlings. The seed- plants. The leaves on the penicillin- lings carrying leaf curl did not improve treated plants (fig. 3, GP) were larger with tetracycline treatment either (Bovt, than those from untreated plants (fig.