UC Riverside International Organization of Citrus Virologists Conference Proceedings (1957-2010)
Journal International Organization of Citrus Virologists Conference Proceedings (1957-2010), 8(8)
Authors Aubert, B. Bové, J. M.
Publication Date 1980
eScholarship.org Powered by the California Digital Library University of California Effect of Penicillin or Tetracycline Injections of Citrus Trees Affected by Greening Disease Under Field Conditions in Reunion Island
B. Aubert and J. M. Bove'
In a companion paper (BovC et al., 1980) For the purpose of antibiotic experi- we explained why the prokaryote asso- ments, the orchard was taken over by ciated with citrus greening disease is not Institut de Recherches sur les Fruits et a mycoplasma, and showed that green- Agrumes (IRFA) and has received ade- ing-affected citrus seedlings, which had quate care since. absorbed penicillin through their roots, The sweet orange varieties, grafted on developed an abundant root system and Troyer citrange and planted 8 by 8 m, produced new, vigorous, symptomless were Valencia late, Hamlin, sweet seed- shoots. On the basis of these results, as ling, Pineapple, and Washington navel. well as previous ultrastructural studies The budwood came from Corsica and (Garnier and BovC, 1977) we concluded was free of known virus and virus-like that the greening organism probably diseases. In 1977, six years after plant- possesses a peptidoglycan-containing ing, most trees showed severe symptoms cell wall typical of gram negative organ- of greening. Yearly outbreaks of the isms and is a member of the division oriental psylla were noted at each flush. Gracilicutes (BovC et al., 1980). In this The presence of the greening organism paper, we add further evidence to this was confirmed by electron microscopy conclusion by showing that, under of leaf samples and graft transmission orchard conditions, penicillin injections of the disease to healthy Madam Vinous into trunks of greening-affected sweet seedlings kept in insect-proof screen- orange- trees result in remarkable tree houses. recoveries similar to those achieved Intensity of greening symptoms. The with tetracycline injections. severity of greening was rated on a scale from 0 to 40, obtained by inspecting the MATERIALS AND METHODS upper and lower halves of the tree on all Sweet orange trees. Trees injected four quadrants and grading each from 0 were in a 1 ha orchard planted in 1971 (apparently healthy) to 5 (severe symp- on well drained, flat, sandy soil, located toms including extensive dieback) and on the leeward side of the island where adding the eight resulting grades. the oriental psylla (Diaphorina citri Injections. In December 1977, three (Kuway.), is prevalent (altitude: 10m) months after bloom, 12 groups of three (Etienne and Aubert, 1980). The annual orange trees were selected in the orchard rainfall is 900 mm and irrigation is ap- for injection. The trees of a given trio plied from September to December. were of the same variety, of similar size, The orchard is kept under permanent and showed greening symptoms of simi- sod; weeds are controlled under the lar intensity (fig. 1A). There were five trees by hand or with herbicides. Two trios of Valencia late, two of Hamlin, kg of a 15-7-24 NPK mix per tree are two of sweet seedling, two of Pineapple, applied annually. Soil analysis has and one of Washington navel (fig. 1). shown that most elements are at the One tree in each trio was injected with optimum level, except magnesium, water (C), a second with penicillin G (P) which is in excess. The orchard was and a third with tetracycline-HCI (T). under private management until 1977 Trees other than those in the 12 trios and relatively poorly kept because of were used for preliminary injection the severity of greening and low yields. experiments. 104 Eighrh IOCV Conference The device for injecting trees is Bioassays. The presence of antibiotics sketched in figure 2 and was developed in the trees as a function of time after by one of us (B.A.). It has been described injection was determined by bioassays elsewhere (BovC, 1978; Aubert, 1979). on Bacillus subtilis (ATCC 6633) for Two hard plastic 1.5-liter bottles were tetracycline, and on Sarcina lutea used per tree. Each bottle was filled with (ATCC 934) for penicillin according to 1 liter of liquid and pressurized to 5.0 the method of Grove and Randall kg/cm2before connecting it to the tree. (1955). The assays were performed on The 36 trees were each injected with the 200 mg (fresh weight) of leaf midribs, 2 liters of liquid over a two-day period. flowers, or rootlets, taken at random Uptake time averaged 1 hour 45 min- from the injected trees. Rootlets were utes with a range of 20 minutes to 4 surface sterilized in 1 per cent sodium hours. The .first injection was carried hypochlorite for 10 seconds and rinsed out in December 1977 with water, peni- three times in sterile water. After grind- cillin G or tetracycline-HC1. The anti- ing the sample with 1 ml of water in a biotics were applied at 3 g/liter (6 mortar, 20 p1 of the homogenate was g/ tree). Only penicillin G was injected deposited on a 6-mm Whatman AA or in June and September 1978 by the Schleicher-Schull 2668 paper disk. For same methods, for a total of 18 g/tree fruit near maturity the bioassays were compared to 6 g/tree for tetracycline- made directly with 20 p1 of fruit juice. HCI. The disks were placed on freshly inocu- The severity of greening symptoms lated culture medium. The diameters of was recorded in October 1977, before the inhibition zones were read after 24 the injections, and in October 1978. hours at 28O C and compared with those Yields of individual trees were deter- given by known amounts of penicillin G mined in March-April, 1978 and 1979. or tetracycline-HCI.
I '. o.val~nr.ra Late namlin I 11 Ill IV v VI VII Fig. 1. Symptom intensity (A) and yield in kg/tree (B) of water, penicillin, or tetracycline injected sweet orange trees. Greening, Stubborn and Related Diseases
Fig. 2. Device for tree injection. Fig. 3. Relation between penicillin concen- tration, X and diameter of inhibition zone, Y.
The relation between penicillin co?- minutes for the liquid to enter the trees. centration, X, given in international A sixth tree received a paste of 15 g of units (1 IU = 0.607 pg) and diameter of dry penicillin in vaseline applied to six inhibition zone, y in cm, was found to holes drilled in the trunk. Penicillin in be log y = 4 x + 1 by the least squares the leaf midribs was estimated at method (fig. 3). various times after injection (fig. 4). No Both penicillin G and tetracycline- antibiotic could be detected at any time HCl were obtained from SPECIA. when trees were injected with water or when penicillin was applied in a paste. RESULTS However, penicillin could be detected in Bioassays. No inhibition zone was ob- the leaves at its highest concentration as served in any tissues of the 12 trees in- early as I hour after the beginning of jected with water. Since this work was injection, when injected with a solution essentially initiated to study the effect of containing 6,9, or 12 g of the compound. penicillin, more bioassays were con- With 3 g, the highest concentration was ducted with leaf midribs from penicillin- detected 10 hours after injection. Penicil- injected trees than those from tetracy- lin remained at a high level for approxi- cline-treated trees. The main purpose of mately 70 hours, then decreased, and the bioassays was to determine the up- could not be detected after 120 to 145 take, distribution, and persistence of the hours. In small stunted trees, penicillin antibiotic in the tree. Inhibition zones (6 gltree) was still present after 250 to penicillin or tetracycline could be hours. seen with extracts from young leaf mid- The antibiotic concentration in ribs harvested as early as 15 minutes flowers followed approximately the after injection was begun. In one experi- same pattern as in leaves, but it was ment, five Washington navel trees were always 20 per cent lower in fruit juice. injected with 0, 3, 6, 9, or 12 g of pen- Penicillin could be demonstrated in the icillin in a volume of 2 liters. It took 20 roots of some, but not all, trees. Eighth IOCV Conference
Effect of penicillin and tetracycline of penicillin is less persistent than that on symptom expression and yields. of tetracycline and repeated penicillin Trees of trios I, 11, V and X had the most treatments were required. Indeed, in severe symptoms of greening with a May 1979 some of the penicillin-treated rating of about 30 (fig. lA), and had trees have begun to show leaf symptoms very low yields (fig. 1B). Within these of greening again. This confirms results trios, trees injected with water (C) obtained in the greenhouse where severe continued to show severe symptoms symptoms of greening reappeared soon (fig. 1A, September 1978 observations) after penicillin was no longer applied and their yields remained low (fig. IB, (BovC et al., 1980). April 1979 harvest). In contrast, trees The tetracycline was injected at 6 injected with penicillin (P) or tetracy- g/tree. Tree weight, determined after cline (T) showed a remarkable recovery, uprooting, was approximately 250 kg. produced many virogous new shoots With such trees, injection of 6 of tetra- with large expanded leaves, and symp- cycline had a phytotoxic effect within tom intensity decreased to a rating of four months after treatment. and re- about 5 (fig. 1A). Yields in April 1979 sulted in a general yellowing of the were greatly increased over those of foliage and a brown discoloration on April 1978 (fig. 1 B). the margins of new leaves. Later, the The trees of other trios were less phytotixic effect disappeared and severely affected (grades around 10) and vigorous new shoots with large, normal- their yields were not as low. Water- colored leaves were produced. No phy- injected control trees had higher yields totoxicity was observed from penicillin. in general in April 1979 than in 1978, DISCUSSION which reflected the improved orchard care since 1977. The penicillin or tetracy- Penicillin has a marked effect on cline-injected trees produced vigorous, greening-affected trees, inducing the symptomless shoots and many trees formation of new, vigorous, symptom- appeared almost normal (grades below less growth. Fruit quality is improved 5). They had much less fruit drop in and yields are significantly increased. December 1978. Their 1979 yields were The results obtained under field condi- far higher than those of 1978, and were tions with penicillin injected in the also higher than the 1979 production of trunks confirm and extend those ob- the water-injected control trees. tained in the greenhouse where penicil- In summary, for the 12 trios the aver- lin was fed through the roots of young, age 1979 production of the water-, greening-affected, sweet orange seed- penicillin-, and tetracycline-injected lings (BovC et al., 1980). As discussed trees was 33, 76, and 99 kg/ tree, re- elsewhere (Bovt et al., 1980) on the basis spectively. In addition to low yields, the of these data as well as on ultrastruc- water-injected trees had many small, tural studies in situ (Garnier and BovC, lopsided fruits. Although the penicillin- 1977) the greening organism definitely injected trees were markedly improved is not a mycoplasma, but a prokaryote in comparison with the water-injected probably having a peptidoglycan-con- controls, they remained inferior to the taining gram negative type of cell wall tetracycline-injected trees except in (Gracilicutes; see Gibbons and Murray, trios 11, VII, VIII, and XI1 where the 1978). penicillin-treated trees had the highest Both penicillin and tetracycline are yields. effective against greening. A single appli- cation of 6 g tetracycline per tree has a The penicillin-treated trees have re- longer lasting effect than the same ceived three injections totaling 18 g amount of penicillin. At this rate, tetra- (December 1977, June, and September cycline is phytotoxic, but penicillin is 1978)' while the tetracycline-treated not. trees were injected only once with 6 g (December 1977). The beneficial effect Greening Stubborn and Related Diseases f Penicillin per
o 15g penicillin paste
1 10 30 60 75 120 145 time after inject ion c hrs)
Fig. 4. Penicillin concentration in leaf midribs as a function of time after injection. Eighth IOC V Conference LITERATURE CITED AUBERT, B. 1979. Une technique de'injection sous pression dans le tronc des arbres. Fruits 34: 195-98. BOVJ?, J. M. 197:. Traitment de la maladie du greening des agrumes. Brevet no 780785-5 depose le 17 mars 1978. BOVE, J. M., P. BONNET, M. GARNIER, and B. AUBERT 1980. Penicillin and tetracycline treatments of greening disease-affected citrus plants in the glasshouse, and the bacterial nature of the procaryote associated with greening, p. 102-07 this volume. ETIENNE, J., and B. AUBERT 1980. Biological control of psyllid vectors of greening disease on Reunion Island, pl 118-21 this volume. GARNIER, M:, and J. M. BOV~ 1977. Structure trilamellaire des deux membranes yui entourent les organismes procaryotes associks a la maladie du "greening" des agrumes. Fruits 32: 749-52. GIBBONS, N. E., and R. G. E. MURRAY 1978. Proposals concerning the higher taxa of bacteria. Int. J. Syst. Bacterial. 28: 1-6. GROVE, D. C., and W. A. RANDALL 1955. Assay methods of antibiotics. Antibiotics Monographs No. 2: 1-238. Medical Encyclopedia, Inc.. New York.