Proc. Fla. State Hort. Soc. 91: 243-245. 1978.

PRELIMINARY STUDIES ON LETHAL PALM BLIGHT

James K. Dunaway and Dana G. Mead not on the Lethal Yellow list. An estimated sixty % of Little Green Acres, Inc., the mature Malayan coconut population now is dead in 21200. S.W. 177 Avenue, Miami, FL 33187 South . Young Malayan palms were found to be more resistant than older Malayan palms in laboratory Abstract. In recent years many additional new species of tests. palm trees in South Florida have been added to the Lethal The implication here may be that resistance to the Yellow list. A large number of palms heretofore thought disease is proportional to the age ofthe 's cell tissue. resistant to the disease, e.g. the 'Malayan Dwarf/ have also This would seem plausible because monocotyledonous been dying by the thousands. tend to retain older cells throughout the life of the During this study Pseudomonas aeruginosa was isolated plant as well as adding new growing cells. This is in con repeatedly from diseased tissue. The disease and lethal de trast to dicotyledonous plants, which are continually re cline of the majority of coconut palms in Florida appears placing older cells with new ones. This may indicate to be initiated by numerous factors, many of which must that disease resistance is linked to plant cell senescence. occur in sequence to causedeath of the tree. In addition to Malayan palms, infected and dying Roy Preliminary results indicate that adequate control of stonea regia (Royal palm), Thrinax argentata (Key palm), this disease syndrome in palms is possible by application of Sabal palm (Cabbage palm) were examined. These palms chemicals. are all reported to be resistant to Lethal Yellow. Coconut palms, injected with oxytetracycline HC1, were seen bleeding and oozing. P. aeruginosa was isolated from The most devastating epidemic in the history of Florida the injection site, cultured in vitro and transmitted and horticulture has been affecting coconut palm trees in South recovered in pure culture from field and tissue cultured Florida since1971. The first lethal yellowing attacks were plants. reported in Jamaica in 1891 and by 1944, the mortality rate It is well known that bacteria including P. aeruginosa near Montego Bay was estimated at 200,000 trees per year. that are treated with antibiotics acquire resistance to those Since the epidemic outbreak in South Florida, at least antibiotics in vivo and in vitro. As there have been over 500,000 palms have been killed. These are valued at over one million injections of palms with oxytetracycline HC1 one hundred million dollars. Coconuts observed in Mo in Florida, it is possible that the once resistant Malayan zambique, Mexico, the , Southeast Asia, Hono coconuts have been infected with an antibiotic-resistant lulu and other coconut producing areas of the world have strain of the bacterium. It is possible that a mutant has been reported to exhibit similar symptoms. These symp developed that is capable of causing the Lethal Yellow syn toms reported from such widely separated areas may be drome. caused by the same organism. Some examples would be, bud rot (Phytophthora palmivora), Heart rot (flagellates), Red ring (nematodes), Lethal Yellow (Mycoplasma), and Materials and Methods Bronze Wilt. Twenty four other species (Table 1) of plants in South Several samples of tissue were taken from each affected Florida currently are showing lethal blight symptoms coconut tree and taken to the laboratory. Obvious trauma similar to those seen in coconut palms and are not included to the trees due to recentcold damage, gross infection, and/or insect damage was recorded. in the official list. The coconut tree samples were trimmed into serial Table L Species of plants in South Florida showing Lethal symptoms sections of tissue and further examined for any indications similar to Palm Yellows. of pathology. Tissue sections were examined in wet mount with a dissecting microscope. Any damage to tissue or

Scientific name Common name organisms found were recorded. Samples of tissue were prepared using standard tissue preparation techniques in

Chamaerops humilis European fan palm a sterile hood. The tissue selected for inspection was Chrysalidocarpus spp. excised from the internal advancing edge of necrosis. Coccothrinax crinita Grand Dad Palm Further optical examination was done. Fungi, mites Copernicia alba and nematodes, when present were photographed. Sections Copernicia bertoriana Cyperus papyrus Papyrus reed of tissue examined include bud sections, leaves including Dracaena draco Devil tree stomata and vascular system, roots, root hairs, trunk and Howeia fosteriana Kentia palm necrosing inflorescence. Staining and cultural procedures Cocos nucifera Malayan dwarf, Jamaican tall for bacteria and fungi were done according to standard Musa nana Cavendish banana utilis Screw published microbiological methods. Identification was Persea americana Avocado effected by use of Roche Diagnostic and Analtab Inc. Rhapis humilis Lady palm systems, supplemented by other techniques. Roystonea regia Royal palm Nematodes and mites were grown on coconut tissue Sabal palmetto Cabbage palm Thrinax argentata Silver palm media. Further identification was doneby the University of Thrinax microcarpa Key palm Florida in Gainesville. Thrinax paraviflora Key palm Mycoplasma-like organisms (MLO) were diligently Washingtonia robusta sought by cultural methods. Thin sections were stained and Washingtonia filifera examined for these organisms. Clinical Mycoplasma indi cator media were used in addition to commercial media Malayan coconuts were formerly considered resistantto specifically for MLO. Lethal Yellow blight. Thus this paper will discuss pri After the etiological agent was identified experiments marily the Malayan Coconut and some of the other palms were designed and executed to determine if the isolated

Proc. Fla. State Hort. Soc. 91: 1978. 243 P. aeruginosa was associated with the palm disease symp negative rod, motile, with 3 color polar flagella. The toms, e.g. yellow or brown flagging and necrotic buds. The colonies exhibited green fluorescence indicative of the experiments described demonstrate that such a relationship Fluorescens group. is present. At first we expected to find P. marginalis or Fluorescens In the laboratory, the isolate was found to infect plant Bio Type II. The bacteria demonstrated substantial growth tissue readily. Sterile coconut heart tissue was inoculated at 42 °C and abundant blue green pyocanin pigment on with pure culture of P. aeruginosa. Within 24 hr the sterile Difco isolation media. tissue was rendered into a brown, fetid smelling, mucoid We were curious about a possible synergistic action, but liquid. The only organism recovered from this culture the comparison results with pure culture showed that no after liquefaction was P. aeruginosa. These experiments such action was necessary. Apparently it does not produce were repeated with P. aeruginosa and Serratia marcescens any discernible pectolytic enzymes. However, it actively (in mixed culture), P. aeruginosa and Erwinia spp. (in decays cellulose and other plant tissue. mixed culture), and also P. aeruginosa and P. marginalis P. aeruginosa was inoculated into coconut tissue simul or Fluorescens Bio Type II. A special inoculation series taneously with Phytophthora palmivora. It was noted that of P. aeruginosa and Phytophthora palmivora was done in the Phytophthora did not compete well and soon deterio vitro to explore any possible interactions. rated. When Phytophthora palmivora was inoculated first, A media consisting almost entirely of sterile autoclaved approximately one week ahead of P. aeruginosa, the effect apical region tissue was prepared. This pulpy mixture was was identical. If P. aeruginosa was inoculated first, then then inoculated with ordinary cultures of P. aeruginosa, S. Phytophthora palmivora never grew at all. Apparently the marcescens, P. marginalis, P. fluorescens Bio Type II, Phy metabolic by-products from P. aeruginosa were not com tophthora palmivora, and P. aeruginosa cultured on coconut patible with a good growth of Phytophthora palmivora. tissue for thirty days previously. The only flask showing any Although P. aeruginosa is not usually considered to be an notable reaction was the coconut cultured P. aeruginosa. invasive primary plant pathogen, it did digest coconut Overnight it began to foam the culture and caused an ex tissue in vitro in 48 hr. In the first field test, series reactions treme liquefaction of the culture media. were observed in 3 weeks in the form of yellow flagging and After 10 days at 27 °C, the cultures were examined and brown wilting. All trees but the control succumbed to the the only culture which showed any extreme liquefaction inoculum. Some speculation was voiced about the Thio was the P. aeruginosa cultured previously on coconut tissue. medium used in the inoculum perhaps being phytotoxic. This culture of P. aeruginosa was then processed in a Giving due consideration to such a suggestion, a new tree search for an enzyme which might explain the exceptional was selected and a control of Thio media without P. virulence of this particular culture. If one agitated the aeruginosa was prepared, at present no ill effects have flask, the entire culture turned from the normal creamy come from the medium. However all subsequent transmis yellow color to a sooty, aqua blue color. This enormous sions were carried out using aqueuos solutions of inoculum. concentration of pyocyanin dye was not found in the other It was observed in the laboratory that some cultures cultures. All cultures were then centrifuged and put under of P. aeruginosa caused marked phytotoxic effects in only a low pressure vacuum filter. The resultant fluids were a few hours. In many cases the tissues were seriously then transferred to be tested on in vitro material. afflicted and browned over night. This unexpected result The first and most reliable method of in vitro trans gave rise to some theorizing about toxic effect and why mission was to use sterile cultured plant tissue. The families some only induced a necrotic effect. The burned appear of plants used were Araceae, Rutaceae, Solanaceae, Palmae, ance of the in vitro tissue lead the investigators to consider , Leguminosae, Cactaceae, Lauraceae, Brome- an enzymatic action of some type and this could have been liaceae and Musaceae. the source of the phytotoxic reaction. The notable results A second method of testing was to use small tree fronds were the tubes containing the P. aeruginosa, which were and leaflets of large palms. The leaflets and fronds selected cultured exclusively on coconut tissue. Overnight the vascu were healthy, well-kept and surface sterilized. They were lar system of the plant apparently picked up a filterable inserted into small tubes and capped with orchid stoppers enzyme or toxin and began to translocate it throughout or spun Dacron. Subsequently the various nitrates were the entire leaf or stem. The effects in palms were shown as a added to the tubes containing the tissue. light browning of the phloem tubes and general classic The production of acceptable symptoms in the field appearing chlorosis. This appearance was inevitably presented many problems. Such variables would include, followed by total browning and wilting. pre-infection, general tree health, environment and the The leaflets and fronds, which gave the most spectacu omni-present possibility of secondary or simultaneous in lar results, were from coconut palms, Jamaican tall and fection. To help eliminate many variables in vitro trans Malayan Dwarf (Cocos nucifera), Kentia palm (Howeia mitting was initiated simultaneously. fosteriana), and Fishtail palm (Caryota mitus). The filtrate In early June of 1978, 6 coconut trees were carefully was then tried on plant tissue in vitro. The results from selected in a field nursery in south Dade County and the the filtrate were far more effective and immediate than trees were inoculated with P. aeruginosa. One tree was used using P. aeruginosa cultured on standard Bacto agars. as a control and inoculated with sterile water. In late None of the plants tested could withstand the effects of August, 1978 a new field inoculation series was then imple the filtrate tested. The cell-free filtrate is now being analyzed. mented and is currently in progress. A total of 33 coconut It is apparently a powerful phytotoxin and can easily cause trees were selected in the same field nursery. Many of the severe chlorosis in plants in dilute solution. trees were blooming or bearing fruit, and all were in very The active agent is filterable through 0.2 micron milli- robust condition. At this time the tests are still in pro pore filter. It is heat labile, and apparently easily trans gress with positive results. ported through the plants vascular system.

Results and Discussions Summary

P. aeruginosa was isolated from 99% of all the un P. aeruginosa was consistently isolated from unhealthy healthy tissue samples examined. It appears as a Gram tissue. Several species of fungi were also isolated. The most

244 Proc. Fla. State Hort. Soc. 91: 1978. notable were rare cases of Phytophthora infestans and/or is possible. Approximately 800 coconut trees are presently palmivora. The host trees usually had badly rotted buds. involved in various forms of therapy. The environmental Other fungal species usually found on the exterior of the conditions are kept as stable as possible in a well-kept trees were of the genera Aspergillus, Penicillium and Fusar- field nursery. At this time preliminary results seem to offer ium. At no time were Mycoplasma isolated. a 99% remission of symptoms. Many of the test specimens Bacteria isolated included S. marsecens, P. fluorescens are in bloom or in fruit. Bio Type II, Erwinia spp. and Citrobacter spp. Most Work now in progress is attempting to define what may numerous and most common was P. aeruginosa. P. be a mutative genome of the etiological agent. It has in aeruginosa appeared in the examined tissue with predictible fected a variety of host representing many families. The consistency, in contrast to other micro-organisms. mutative effect of broad spectrum antibiotics on standard At the present time, efforts are continuing in the areas cultures of P. aeruginosa is being studied. P. aeruginosa is of vector analysis and blight control. Preliminary test re a recognized human pathogen. sults indicate that a course of therapy and/or prevention

Proc. Fla. State Hort. Soc. 91: 245-247. 1978.

BACTERIAL BLIGHT OF FISHTAIL PALM, A NEW DISEASE

J. F. Knauss The research reported herein details the description and IF AS Agricultural Research Center, etiology of the disease, the identification, proof of patho University of Florida, genicity, and suggestions for control of the causal patho Rt. 3, Box 580, Apopka, FL 32703 gen.

AND

J. W. Miller and R.J. Virgona Materials and Methods FDACS, Division of Plant Industry, Isolations from diseased leaves were made from ad P. O. Box 1269, Gainesville, FL 32602 vancing margins of active lesions employing the technique detailed earlier (3). Cultures of the characteristic bacterium Additional index words, foliage plants, Pseudomonas, consistently isolated from the diseased palm leaves were Caryota. submitted to various physiological and pathological tests (1) to determine their identity. Abstract. Diseases of palms caused by bacterial patho Four isolates of the suspect bacterium were selected to gens previously have not been encountered in the Florida test their pathogenicity to healthy plants of C. mitis. The tropical foliage plant industry. In the summer of 1977, a inoculum for each isolate was prepared by transferring a severe brown to black foliar blight was found uniformity loop full of a 24-hr-old lima bean agar tube culture grown affecting a large percentage of young fishtail palm plants at 80F dz 2 (27C ± 1) to a flask containing sterile yeast imported into central Florida from a south Florida source. extract + dextrose broth (10 g each/1). The flasks were Cultural isolations of diseased tissue consistently produced a incubated on a reciprocal shaker at 80F dz 2. After 6 hr the bacterium identified as Pseudomonas alboprecipitans. Com contents of the flasks were individually spun down in sterile pletion of Koch's Postulates on proof of pathogenicity indi centrifuge tubes for 15 min, the supernatant then poured cates this bacterium to be the causal agent of the foliar off, and the bacterial pellet resuspended to the original blight of fishtail palm. volume with sterile distilled water. All succeeding inocula tions were performed in a glass greenhouse with tempera Members of the Palmae are an important segment of ture of 65-90F (18-32C). Florida's ornamental tropical foliage plant industry. Palms, Approximately 2-year-old plants were inoculated, 4/ especially the larger types and including fishtail, are usually isolate, with 4 additional plants serving as controls. All started from seed and grown to mature size in south Florida plants were placed under mist (15 sec every 15 min) for under saran-covered structures where foliar wetting from 24 hr prior to inoculation. Thirty ml of inoculum/isolate overhead irrigation and rain is normal. These conditions was applied with an atomizer to the upper and lower sur are ideal for the development of foliar disease problems, a faces of the leaves of the appropriate group of plants. The common occurrence within the Palmae (4, 6). Of all the control plants were treated similarly but received 30 ml reported foliar pathogens of palms in Florida, not one is a of sterile distilled water. After inoculation, wet paper towel bacterium (6). Bacterial pathogens of palms are a rare ing was placed upon the soil surface of each pot, and the occurrence throughout the world, one being Xanthomonas plant and pot covered with a polyethylene bag (secured vasculorum (Cobb) Dowson on Areca palm (4, 5). with a rubber band) and placed under mist (15 sec every In the summer of 1977, a severe blight of Caryota 15 min). After 7 days the mist was terminated, the bag re mitis Lour., fishtail palm, was brought to the attention of moved, and reisolations performed from the diseased leaves the senior author. The plants originally were obtained from of each plant. Two reisolates from each suspect culture a foliage nursery in south Florida. Isolations from diseased tested were further evaluated by physiological and patho tissue consistently yielded a characteristic bacterial organ logical methods to determine if they were identical to the original culture employed for inoculation. ism. Prior to inoculation, the leaves were marked to deter

iFlorida Agricultural Experiment Stations Journal Series No. 1485 mine the effect of maturity upon susceptibility to the patho and Contribution No. 455, Bureau of Plant Pathology. gen.

Proc. Fla. State Hort. Soc. 91: 1978. 245