68 FLORIDA STATE HORTICULTURAL SOCIETY, 1967

No attempt was made to compare vigor and free of the common citrus viruses. In 1965 co tree size between varieties, because any lack of operative arrangementns were made with releas vigor due to xyloporosis was masked by severe ing agencies and theCitrus Budwood Registra cold damage from freezes in 1957 and 1962. tion Section to initiate indexing of new varieties However, there were trees of all three varieties prior to release as a safeguard against possible in the portion containing Temple and Parson seed transmission of viruses. Brown interstocks that appeared to be suffering It is important that contamination with the effects of xyloporosis as well as damage from xyloporosis virus be prevented and this may be freezing. assured only by propagation of material from the official releasing agency on previously un- Results budded rootstocks. In 1965 the Florida Department of Agricul Ten bark specimens examined from the Or ture in cooperation with releasing agenciesbe lando and Robinson on the Valencia interstocks gan a "Validated Source Planting" program. revealed no symptoms, while all except two Through this program interested growers and specimens (Robinson) were infected in the nurserymen are assisted in establishing sources blocks previously containing Temple or Parson of authentic material from new variety releases. Brown. Thus the virus appears to have been Material to be approved must be handled in present prior to the topworking, either in the ways designed to prevent virus contamination. Temple or Parson Brown, or both. Average se The program has met with wide acceptance, and verity ratings for symptom expression were: suppliesof "validated" material are rapidly be Orlando 4.2; Robinson 2.0; Lee 2.4. These ob coming available within the industry. The pros servations indicate Robinson and Lee are less pective grower of these new hybrids should set severly affected than Orlando; and Robinson tle for nothing less than "validated" propaga is only slightly less sensitive than Lee in propa tions. gations of the same approximate age.

LITERATURE CITED Discussion 1. Childs, J. F. L., 1951. Cachexia, a bud-transmitted disease, and the manifestations of phloem symptoms in While Robinson and Lee do not appear as certain varieties of citrus, citrus relatives, and their hybrids. Proc. Florida StateHort. Soc. 64: 47-51. severely affected as Orlando, it should be em 2. Childs, J. F. L. 1952. Cachexia disease, its bud phasized that these propagations are only four transmission and relation to xyloporosis and to tristeza. Phytopathology 42: 265-268. years old at this time. Because xyloporosis may 3. Childs, J. F. L., G. R. Grimm, T. J. Grant, L. C. Knorr and G.G. Norman. 1955. The incidence of xylo not show severe effects at this early age, it may porosis (cachexia) in certain Florida citrus varieties. Proc. be some years before the true pattern will be Florida State Hort. Soc. 68: 76-82. 4. Childs, J. F. L. 1956. Transmission experiments and known. xyloporosis-cachexia relations in Florida. Plant Dis. Reptr. Vol. 40: 143-145. Two additional varieties, Osceola tangerine 5. DuCharme, E. P. 1951. Xyloporosis of citrus. Proc. and Nova tangelo, are products of the same par Florida State Hort. Soc. 64: 57-60. 6. Norman, G. G. 1958. Florida State Plant Board ents that produced Robinson and Lee and could Program for virus-free budwood. Proc. Caribbean Region Amer. Soc. Hort. Sci. pp. 1-7. be similarly affected. All of these new hybrids 7. Riechert, I. J. Perlberger. 1934. Xyloporosis, the new are of seedling origin and have been presumed citrus disease. Bull. 12. The Jewish Agency for Palestine, Agr. Exp. Sta. Rehoboth 1-50.

EXOCORTIS VIRUS OF CITRUS CAN BE SPREAD BY CONTAMINATED TOOLS

S. M. Garnsey Abstract

Crops Research Service Exocortis virus of citrus was transmitted at Agricultural Research Service a high rate between citron plants with contami U. S. Department of Agriculture nated grafting tools. Orange and grapefruit Orlando seedlings were less susceptible to infection by GARNSEY: EXOCORTIS 69 this means. The virus remained infectious for This paper presents evidence that exocortis 16 hours on a knife blade; however, contami virus can be mechanically transmitted with con nated tools were sterilized by dipping in a mix taminated tools and discusses the practical im ture of 2% sodium hydroxide and 2% formalde plications of this discovery for the nurseryman hyde. Caution is recommended in nursery op and grower. erations to avoid accidental infection when propagating trees on exocortis-sensitive root- stocks. New plantings on sensitive rootstocks Methods and Materials should be planned to minimize transmission dur The experiments were conducted in a ing subsequent horticultural operations such as screened greenhouse that was cooled with evapo hedging. rative coolers. Air temperatures usually ranged from 70 to near 90° F, but occasionally reached Introduction 95° F for short periods. The greenhouse was partially shaded with paint to provide a light Exocortis-infected orange (Citrus sinensis intensity of 1500 to 2000 ft-c near bench level L.) 1, grapefruit (C. paradisi Macf.) and tan at midday. All plants were grown in steam- gerine (C. reticulata Blanco) trees on tolerant sterilized potting soil. stocks do not show symptoms. However, propa The exocortis virus cultures used in these gation of infected budwood of these varieties on tests were obtained from citron plants that be trifoliate orange [Poncirus trifoliata (L.) Raf.]1, came naturally infected while growing in the various citranges [P. trifoliata X C. sinensis (L.) greenhouse. They produce typical exocortis Osbeck] and 'Rangpur' "lime" (C. reticulata symptoms (1, 2) in the citron indicators used. var. ouster a) results in stunted trees, which The citron indicators were propagated from often show conspicuous bark-scaling symptoms selected seedlings of Etrog citron and show con on the stock. Exocortis-free budwood is a pre spicuous leaf and stem symptoms (Fig. 2) requisite for successful use of these rootstocks, within 3 months following bud-inoculation with and budwood-source trees must be indexed on a the exocortis cultures above. The Arizona 861, sensitive indicator for exocortis virus. OES-7 and OES-9 selections have been de Indexing procedures for exocortis originally scribed previously (2, 3). The indicators were required 4 to 8 years. A series of refinements, propagated as rooted cuttings or by inserting culminated in the development of the 'Etrog' chip buds on healthy seedlings of other citrus citron (C. medica L.) test (1), which can be varieties as indicated. completed within 6 months. In some tests, inoculations were made by Use of Etrog citron plants in indexing using a contaminated knife in the budding proc studies in the greenhouse led to the discovery ess. In others, inoculation cuts were made on that exocortis can be spread by means other the stem of the stock plant above and below a than graft transmission (3). Unexplained in clean indicator bud (Fig. 1). The knife was fections of exocortis were observed in one or contaminated by drawing the blade through a more propagations derived from five citron stem of an exocortis-infected plant, or by apply seedlings and one old-line selection of Etrog ing a drop of an extract made from excortis- citron. These infections occurred over a period infected tissue to the knife blade. Buds were of 15 months and could not be readily attributed wrapped with polyethylene tape and inoculation to either seed transmission or to errors in cuts were wrapped with Stericrepe tape* inoculation. (Beacon & Janis Ltd, London). Several insects and mites, notably citrus All tools were dipped in an aqueous solution white fly [Dialeurodes citri (Ashmead)] and containing 2% sodium hydroxide and 2% for citrus red mite [Panonychus citri (McGregor)], maldehyde prior to their use on each plant to were occasionally present in the greenhouse avoid accidental contaminations. despite frequent spraying. However, both failed to transmit exocortis to citron plants when tested as vectors (3).

*Trade name is used solely to provide specific information. Mention of a trade name does not constitute a warranty of lThe system of citrus nomenclature used in this paper the product by the USDA or an endorsement over other is that of Swingle (6). products not mentioned. 70 FLORIDA STATE HORTICULTURAL SOCIETY, 1967

that had been freshly passed through an infected stem of Etrog citron prior to cutting each bud. Nine of the 10 citron plants which grew from these buds developed symptoms of exocortis within 4 months. Additional experiments were made to con firm these transmissions. A total of 60 citron bud propagations were made in two tests. All propagations of each of the three citrons used were from a single source plant. One-third were made using a knife contaminated with exocortis, and one-third were made using a knife con taminated with exocortis and subsequently dip ped briefly either in 70% ethyl alcohol or in an aqueous mixture of 2% sodium hydroxide and 2% formaldehyde. Tools dipped in the latter solution were rinsed in tap water before use. As a control, one-third of the propagations were made using a sterilized knife. Within 4 months, 17 of the 20 bud propaga tions made with the contaminated knife showed symptoms of exocortis infection (Fig. 2), while the 20 control propagations remained healthy. Dipping the budding knife in alcohol reduced, but did not completely prevent, transmission. Dipping the budding knife in the mixture of sodium hydroxide and formaldehyde prevented transmission. These results are summarized in Table 1. Survival of virus cm a knife blade.—Two tests have been made to measure the length of time that exocortis virus can remain infectious on the surface of a steel knife blade. In the first test, citron indicator buds were cut immediately, 5 minutes, and 10 minutes after contaminating the knife by passing it through infected citron- stem tissue. Transmission occurred at all inter

Fig. 1.—Illustration of "cut stem" inoculation procedure vals. A second test is now in progress, using used in exocortis transmission tests. intervals of 10 minutes, 30 minutes and 16 hours. A) Budding knife, contaminated previously in infected tissue, is making inoculation cots on stock above citron Infection has occurred in four of five plants indicator (B). B) Citron indicator bud on seedling: rootstock being inoculated at the 10 and 30 minute intervals wrapped with polyethylene tape. and in two of five incoulated at the 16-hour C) Inoculation cuts below indicator, wrapped with Steri- crepe tape. interval.

Virus content of infected tissue.—Tests were conducted to measure the amount of exocortis virus present in infected citron tissue. Small Results pieces of young bark and leaf mid-rib tissue Evidence for knife transmission.—Experi were macerated in .01M cold, neutral phosphate ments were begun in February 1966 to deter buffer. The macerate was filtered to remove mine whether exocortis could be spread with cell debris, diluted to the desired volume, and contaminated tools. In a preliminary test, 10 assayed for infectivity by applying a drop to a bud-propagations of Arizona 861 citron were knife blade and making 6 cuts on the stems of made on sour orange seedlings using a knife the Etrog citron cuttings. In the first test, the GARNSEY: EXOCORTIS 71

highest dilution tested was 1:100, and all 10 plants inoculated at that dilution became in fected. No infections were observed in 10 indi cator plants inoculated with buffer alone. In a recent test, infection has occurred in 4 of 10 plants inoculated with a 1:1000 dilution of mac erated leaf mid-rib tissue. Transmission by clippers.—In the previous tests the site of inoculation was covered with a wrapping; however, exocortis was also trans mitted to citron indicator plants through cuts made with contaminated clippers which were left unwrapped. Susceptibility of orange and grapefruit to mechanical inoculation.—The susceptibility of Pineapple orange and Duncan grapefruit seed lings to mechanical inoculation was compared with that of Etrog citron cuttings. The test plants were budded with healthy Arizona 861 indicator buds and inoculated on the stock ap proximately 1 inch above and below the indi cator bud by making three cuts with a contaminated knife (Fig. 1). Ten plants each, of orange, grapefruit and citron, were inocu lated from orange, grapefruit and citron plants containing the same culture of exocortis. Ten propagations of Arizona 861 citron on stocks of each type were left uninoculated as controls (Table 2). Four months after inoculation, 26 of the 30 citron plants inoculated showed unmistakable exocortis symptoms; after 6 months, three of 30 plants inoculated on the orange stock showed symptoms. All the plants inoculated on

Fig. 2.—Two bud propagations of Arizona 861 citron the grapefruit stock remained symptomless, as 18 weeks after budding. Plant A is from a healthy, unin- did the uninoculated controls (Table 2). oculated bud. Plant B is from a healthy bud that was cut with a budding knife contaminated with exocortis. It shows In a supplementary experiment on knife in severe leaf epinasty and stunting characteristic of exo cortis infection in this indicator. oculation, 4 of 10 orange seedlings became in-

Table 1. Transmission rate of exocortis virus between citron plants with contaminated budding tools!/ Treatment Contaminated Tools Treated With Citron Contaminated 2% NaOH + Uninoculated Indicator Tools 70% ETOH 2% formaldehyde Control Arizona 861 17/20 3/5 0/5 0/10

OES-7 4/5 0/5 - 0/5

OES-9 5/5 0/5 0/5 Total 26/30 3/10 0/10 0/20 17 Summary of three experimetits. 72 FLORIDA STATE HORTICULTURAL SOCIETY, 1967

Table 2. Relative susceptibility of Pineapple removed when the tools are used for pruning orange, Duncan grapefruit and Etrog citron or trimming sprouts, but is removed with water plants to mechanical inoculation v;ith exocortis before using a knife for budding, as the chemi virus cals can kill the bud. Further tests are in prog Inoculum Frequency of infection** ress on sterilants that may be suitable for use source* Orange Grapefruit Citron Orange 1/10 0/6*** 10/10 on large field equipment such as hedgers. Since exocortis virus can remain infectious Citron 2/10 0/6 10/10 on a dry knife blade for at least 16 hours, con taminated tools should be decontaminated if Grapefruit 0/10 0/6 7/10 they are to be used again within a several-day None 0/9 0/6 0/10 period. Further tests will be necessary to de * Each host contained same virus culture. termine the maximum length of time the virus ** Results based on observation of symptoms can remain infectious. in Arizona 861 indicator. *** Ten plants were inoculated, but some Orange and grapefruit seedlings are appar indicator buds failed and these plants ently less susceptible to mechanical inoculation were not recorded. with exocortis than citron cuttings. However, these experiments involved only one type of inoculation, using young plants of only two vari fected, when inoculated as in the previous eties and do not yet form an adequate basis experiment using an infected orange seedling for generalization. The longer incubation period as the inoculum source. The 10 control plants encountered with inoculations made to orange for this test remained symptomless after 6 seedlings also makes it difficult to determine the months. The time required for symptoms to final rate of transmission. In any case, neither appear in the two experiments above was aver orange nor grapefruit should be regarded as aged by rootstock. Symptoms appeared in an immune to mechanical inoculation at this time. average of 71 days for those plants inoculated Seven out of 40 orange seedlings, inoculated on the citron rootstock. The range was 36 to in the experiments reported, did become infected. 98 days. Symptoms appeared in an average of Furthermore, circumstantial evidence is accu 170 days for those plants inoculated on the mulating which suggests that spread of exocortis orange rootsock with a range from 118 to 184 in the field by contaminated tools has occurred days. in Florida (personal communication with G. D. Bridges, State Budwood Program, Winter Discussion Haven), and in California (personal communi cation with Dr. E. C. Calavan, University of Exocortis virus was transmitted readily to California, Riverside). In both areas exocortis Etrog citron plants with contaminated knives or infections have been discovered which cannot clippers, and this type of transmission was cer be traced to propagation of infected budwood. tainly responsible for the unexplained exocortis Mechanical inoculation seems the best explana infections of citron plants observed in our green tion at present, as seed transmission of exocortis house. Following the discovery that exocortis has been reported in only one instance (5), and could be transmitted by contaminated tools, pre tests for insect and soil-borne vectors of this cautions were taken to prevent mechanical virus have been negative (3, 4). transmission. No further "natural" infections The transmission of exocortis by contami have occurred, even though we are now working nated tools is of practical significance to the with much larger numbers of citron plants, and citrus nurseryman and grower. It does not pre many are in experiments containing mixed clude the use of exocortis-sensitive rootstocks, populations of healthy and infected plants. but it does mean that suitable precautions should An aqueous mixture of 2% sodium hydrox be taken in propagating and growing trees on ide and 2% formaldehyde was an effective steri- these stocks. lant for contaminated tools. This solution has also proven practical and effective in every-day It is highly important that nurserymen avoid use in the greenhouse. Knives and clippers are contamination of exocortis-free scion-mother routinely dipped between plants when perform trees because accidental infection of these trees ing any cutting operation. The sterilant is not could result in the subsequent propagation of JOHNSON: RUST MITE CONTROL 73 many infected trees. Care in nursery budding not be readily apparent. In one experiment operations is also indicated, although the mag involving 40 Mexican lime plants [C. aurantifolia nitude of the possible damage is less at this (Christm.) Swing.] the author was not able to point. transmit tristeza with a contaminated knife. Growers should recognize the possibility of More experiments are needed for citrus viruses natural spread of exocortis during horticultural other than exocortis. operations such as hedging. New plantings on LITERATURE CITED exocortis-sensitive rootstock should be planned 1. Calavan, E. C, E. F. Frolich, J. B. Carpenter, C. N. to minimize the possibility of contamination, Roistacher and D. W. Christiansen. 1964. Rapid indexing for exocortis of citrus. Phytopathology 54: 1359-1362. and to facilitate the use of decontamination pro 2. Garnsey, S. M., and M. Cohen. 1965. Response of various citron selections to exocortis infection in Florida. cedures, if these become necessary. Proc. Fla. State Hort. Soc. 78: 41-48. The danger of spreading other major citrus 3. Garnsey, S. M., and J. W. Jones. 1967. Mechanical transmission of exocortis virus with contaminated budding viruses with contaminated tools has not been tools. Plant Disease Reporter 51: 410-413. 4. Nigh, E. L., Jr., and R. M. Allen. 1967. Failure of thoroughly investigated. There is little circum nematodes to transmit citrus exocortis virus (Abst.) Phyto stantial evidence to suggest this type of spread pathology 57: 100-101. 5. Salibe, A. A., and S. Moreira. 1963. Seed trans with either psorosis, tristeza, or xyloporosis. mission of exocortis virus. In W. C. Price [ed.], Proc. 3d Conf. Internat. Organization Citrus Virol., University of However, in the case of xyloporosis, the lack of Florida Press, Gainesville. long-term information on registered plantings, 6. Swingle, W. T. 1948. The botany of citrus and its wild relatives of the orange subfamily (family Rutaceae, plus the lack of a rapid indicator plant, provides subfamily aurantioideae). In H. S. Webber and L. D. Batchelor [ed.], The Citrus Industry, Vol. 1, University of a situation where mechanical transmission may California Press, Berkeley.

GC-9160 AND OTHER MATERIALS FOR CITRUS RUST MITE CONTROL

Roger B. Johnson The present commercial status of GC-9160 and the other experimental materials is uncer University of Florida Citrus Experiment Station tain. None of these materials have legal residue Lake Alfred tolerances and none have been approved by the United States Department of Agriculture for Abstract use on citrus.

GC-9160 was as effective as chlorobenzilate against citrus rust mite in 1964 and far superior Introduction to chlorobenzilate in 1965 and 1966. It thus ap A tremendous change in citrus spraying prac pears to be one of the most promising of the tices occurred in Florida when zineb (1958) and 21 new materials tested. Although an optimum chlorobenzilate (1959) were recommended for dosage of GC-9160 was not determined, dosages the control of citrus rust mite. Prior to 1958, if in the range of 2.0 to 4.0 ounces of active in a grower wished to use oil in the summer, he had gredient per 100 gallon appear to be satisfactory to spray twice—first with sulfur or lime-sulfur for optimum control of citrus rust mite. and then with oil. Zineb and chlorobenzilate, NIA-10242 was as effective as GC-9160, but however, could be mixed with oil and for the information supplied by the manufacturer indi first time offered combined control of citrus rust cates that it may be too toxic for the repeated mite, scale insects, spider mites, and even greasy usage that is necessary to control citrus rust spot disease. mite. N-4543 and Hercules 14503 were also Although a good record of rust mite control promising, but need further teting. has been compiled with chlorobenzilate, there have been occasions when control was far too short. A longer-lasting miticide has been needed Florida Agricultural Experiment Stations Journal Serie and numerous experiments have been conducted No. 2808.