CARIBBEAN FOOD CROPS SOCIETY 44 Forty Fourth Annual Meeting 2008

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1 CARIBBEAN FOOD CROPS SOCIETY 44 Forty Fourth Annual Meeting 2008 Miami, Florida, USA Vol. XLIV - Number 2 Continued Poster Session Abstracts With Some Posters Expanded as Full Papers UF MEETING HOST: UNIVERSITY of FLORIDA IFAS

2 2008 Proceedings of the Caribbean Food Crops Society. 44(2): Poster #69 A New Lethal Disease of Syagrus romanzoffiana and Washingtonia robusta in Florida is Caused by Fusarium oxysporum Monica L. Elliott 1 and Elizabeth A. Des Jardin 1 1 Fort Lauderdale Research and Education Center, University of Florida - IF AS, Fort Lauderdale, Florida 33314, USA Corresponding author: melliott@ufl.edu ABSTRACT Syagrus romanzoffiana and Washingtonia robusta are popular landscape ornamental palms grown throughout most of Florida. Since late 2004, we have noted mature specimens in the landscape and juvenile material in nurseries of S. romanzoffiana with symptoms not observed previously for this species. Symptoms are first observed on the oldest leaves, with individual leaves exhibiting chlorosis and necrosis initially only on one side. A distinct reddish-brown stripe is visible on the petiole and rachis, and there is a corresponding discoloration internally. Within 2-3 months after onset of initial symptoms, the entire canopy desiccates and turns brown as if freeze-dried in situ. Similar symptoms were observed on W. robusta beginning in the spring of 2007 in landscapes and a nursery. Fusarium oxysporum was consistently isolated from symptomatic petiole tissue from both palm species. DNA was extracted from single-spore isolates, and a portion of the translation elongation factor 1-alpha (TEF) was amplified using the polymerase chain reaction and the efl and ef2 primers. The resulting amplicons were sequenced. Comparison of the sequences obtained with TEF sequences in the FUSARIUM-ID database ( demonstrated that this F. oxysporum is likely to be a new forma speciales. Koch's postulates were completed using S. romanzoffiana and IV. robusta and F. oxysporum isolates obtained from both palm species. F. oxysporum isolates from S. romanzoffiana were pathogenic on W. robusta, and, likewise, isolates from IV. robusta were pathogenic on S. romanzoffiana. KEYWORDS: Fusarium oxysporum, palm diseases, Syagrus romanzoffiana, Washingtonia robusta INTRODUCTION Syagrus romanzoffiana (queen palm) and Washingtonia robusta (Mexican fan palm) are landscape ornamental palms grown throughout most of Florida. Quickly dying (within 2-3 months), mature queen palms in landscapes across the southern half of Florida were brought to our attention beginning in late In spring 2007, Mexican fan palms were observed with similar symptoms and disease progression, also throughout southern Florida. The symptoms are similar to those associated with Fusarium wilt of other palm species, such as Phoenix canariensis (Simone, 2004). Symptoms are first observed on the oldest leaves, and the disease progresses up through the canopy. Early, individual leaf symptoms include one side of the leaf blade exhibiting chlorosis and 593

3 necrosis (one-sided wilt or death), and a distinct reddish-brown stripe is visible on the petiole or rachis. There is a corresponding internal discoloration. Within 2-3 months after onset of initial symptoms in the oldest leaves, the entire canopy is desiccated and necrotic as if freeze-dried in situ. There is no indication of trunk or root infection. Studies were initiated to determine the etiological agent of this lethal decline. MATERIALS AND METHODS Internal petiole or rachis tissue of symptomatic leaves was obtained by carefully cutting away the petiole or rachis epidermis. Small internal tissue pieces were placed directly on 1/5 strength potato dextrose agar. FusariumASks colonies were consistently isolated from symptomatic tissue. Therefore, such colonies were selected, purified, single-spored and stored for future use. Isolates were identified morphologically by placement on 1.5% water agar with irradiated carnation leaves embedded in the agar surface (CLA). After 2-4 weeks growth at 26 C with 12 hours light, cultures were examined for presence and characteristics of microconidia, mesoconidia, macroconidia and chlamydospores (Leslie and Summerell, 2006). Fusarium isolates were identified molecularly by obtaining fungal DNA and subjecting to a standard polymerase chain reaction (PCR) protocol using the efl and ef2 primers to amplify a portion of the translation elongation factor 1-alpha gene (TEF), a highly informative region for differentiating Fusarium species and formae speciales of F. oxysporum (Geiser et al., 2004). The approximately 690 bp amplicon was viewed using agarose gel electrophoresis, purified, and then used as a template for DNA sequencing by the DNA Sequencing Core Laboratory, ICBR, UF, Gainesville. Sequences were edited and then queried against the FUSARIUM-ID database using the BLAST search tool ( Geiser et al., 2004)). F. oxysporum isolates PLM-140B and PLM-153B were selected for the first pathogenicity experiment, which was conducted with juvenile queen palms (3-4 true leaves) and initiated in April Both isolates were obtained from symptomatic mature queen palms growing in landscapes. Isolates were grown on potato dextrose agar (PDA) at 26 C with 12 hours light. Spore suspensions (10 7 per ml) were made with sterile water. Each palm was inoculated with two methods. First, a 4-mm hole was drilled into the palm 8-10 cm above the soil line (and above the apical meristem), and then a 2 ml spore suspension was slowly injected internally. Second, a 5-cm shallow slit was made on the adaxial surface of the petiole of the second, youngest fully-expanded leaf, and a 1 ml spore suspension was dribbled on this slit. Palms designated as controls were handled in the same manner but with sterile water. Palms were immediately placed in clear, polyethylene bags and placed in an area with no direct sun for 3 days. Bags were removed, and the palms placed in a sun nursery with daily overhead irrigation. There were four replicate palms per treatment. Fusarium oxysporum isolates PLM-140B (queen palm, Broward Co. landscape), PLM-246B (queen palm, Lee Co. field nursery) and PLM-249A (Mexican fan palm, Lee Co. field nursery) were selected for the second pathogenicity experiment, which was conducted with seedling queen and Mexican fan palms (3-4 seedling leaves) and initiated in May Isolates and spore suspensions were prepared as described previously. Palms in conetainers were inoculated by pipeting 40 ml spore suspension between the leaf bases, with excess suspension percolating through the potting mix. Palms designated 603

4 as controls were handled in the same manner but with sterile water. Palms were immediately placed in clear, polyethylene bags and placed in an area with no direct sun for 3 days. Bags were removed, and the palms transplanted into 450-ml pots using a peat moss/sand/perlite (1:1:1) potting mix. Palms were placed in a covered shadehouse and irrigated daily. There were five replicate palms per treatment, and each palm species was inoculated with each fungal isolate. This experiment was repeated in October 2007 using these same three F. oxysporum isolates plus F. proliferatum PLM-137B and F. semitectum PLM-138B. These latter two isolates and F. oxysporum PLM-140B had been isolated from the same symptomatic queen palm. All experimental preparations and conditions were the same as described above. RESULTS AND DISCUSSION The vast majority of the isolates were tentatively identified as Fusarium oxysporum based on morphological characteristics. Macroconidia were formed on CLA in orange sporodochia, and were 3-4 septate with a foot-shaped basal cell and curved apical cell. Microconidia were produced in false heads on short phialides, were unicellular and were primarily oval, elliptical or reniform in shape. Chlamydospores were readily produced by most isolates in less than 4 weeks. The only other Fusarium species isolated were identified as F. proliferatum and F. semitectum. F. proliferatum had macroconidia that were longer and more slender than the F. oxysporum isolates, and microconidia (distinctively club-shaped with flattened base) were present in long chains on polyphialides. F. semitectum was identified based on lack of microconidia production, but production of fusoid-shaped mesoconidia were readily observed in characteristics pairs on polyphialides. Macroconidia were shorter than the F. oxysporum isolates. These two species were not consistently isolated from symptomatic tissue as was F. oxysporum. The vast majority of the Fusarium isolates most closely matched Fusarium oxysporum, but did not match any of the formae speciales TEF sequences currently available in the database, including those for F. o. canariensis, the etiological agent of Fusarium wilt of Phoenix canariensis which is already present in Florida. A phylogenetic tree was developed using representative F. oxysporum isolates from queen and Mexican fan palms and F. o. canariensis (isolates from diseased Phoenix spp. in Florida), with a F. semitectum isolate from a queen palm used as the root (Figure 1). 604

5 PLM-181C-Sr-Martin Co landscape - PLM-256D-Wr-Pinellas Co landscape PLM-179D-Sr-Manatee Co landscapeb PLM-192D-Sr-Orange Co landscape PLM-160C-Sr-Manatee Co landscapea PLM-119C-Sr-Martin Co nursery PLM-199B-Sr-Manatee Co landscapec PLM-258B-Wr-0range Co landscape PLM-246B-Sr-Lee Co nursery PLM-249A-Wr-Lee Co nursery - PLM-140B-Sr-Broward Co landscape PLM-386B-F.o.canariensis PLM-183C-F.o.canariensis PLM-221B-F.o.canariensis PLM-225C-F.o.canariensis PLM-138B-F. semitectum 0.02 Figure 1. Phylogenetic tree of Fusarium oxysporum isolates causing a new, lethal disease of Syagrus romanzoffiana (Sr) and Washingtonia robusta (Wr), compared with Fusarium oxysporum f. sp. canariensis. In the pathogenicity experiment using juvenile queen palms, all control palms were healthy after 9 months, but 2 of 4 inoculated palms of each F. oxysporum isolate treatment had died after exhibiting leaf symptoms typically observed in the landscape. F. oxysporum was isolated from symptomatic tissue. The remaining 2 replicate palms of each pathogen-inoculation treatment were healthy and never exhibited symptoms during the 9-month period. In the pathogenicity experiment using seedling queen and Mexican fan palms, symptoms were observed on Mexican fan palms within 3 weeks of inoculation, and all five replicate palms of all three F. oxysporum treatments were dead after 5 weeks. Controls were healthy. For queen palms, no symptoms were observed until 8-9 weeks after inoculation. By November 2007, all five replicate palms infested with PLM-249A were dead, 3 of 5 replicate palms infested with PLM-246B were dead, and 2 of 5 replicate palms infested with PLM-140B were dead. By January 2008, the remaining inoculated palms had not died, but were severely stunted, had ceased to produce new leaves, and only the youngest leaf or leaves were still green. Controls remained healthy. F. oxysporum was isolated from symptomatic tissue. The repetition of this pathogenicity experiment yielded similar results with the F. oxysporum isolates. All 5 replicate Mexican fan palms of all three isolate treatments were dead after 6 weeks. By 12 weeks, 4 of 5 replicate queen palms of all three isolate treatments were dead and the 5th replicate was declining. All control palms for both species remained healthy, as did the palms inoculated with F. proliferatum and F. semitectum (Figure 2). 605

6 I λ.. v-vt. ι-* α.«. β A ; IV ' Λ..ÂtaÉ s?, lifi ifel ^tt Figure 2. Appearance of Syagrus romanzoffiana at 10 weeks after inoculation with Fusarium isolates. Top row: Control, Fusarium proliferaturm PLM-137B, Fusarium semitectum PLM-138B. Bottom row: Fusarium oxysporum PLM-246A, Fusarium oxysporum PLM-249A. Based on the results obtained to date, we believe a new forma speciales of Fusarium oxysporum is the etiological agent of a new lethal disease of queen and Mexican fan palms occurring in Florida. While the disease is primarily observed in mature landscapes, where palms have been established for 5 or more years, it has been observed in three nurseries, one was a container nursery and two were field nurseries with juvenile palms. Because of the wide geographic range of the disease in Florida, wind movement of the pathogen is implicated. REFERENCES Geiser, D. M., Jimenez-Gasco, M. M., Kang, S., Makalowska, I., Veeraraghavan, N., Ward, T. J., Zhang, N., Kuldau, G. Α., and O'Donnell, K FUSARIUM-ID v. 1.0: A DNA scqucncc database for identifying Fusarium. European Journal of Plant Pathology 110: Leslie, J. F. and Summerell, Β. Α The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA. \ - 606

7 Simone, G. W Fusarium wilt, pp , Compendium of ornamental palm diseases and disorders, M. L. Elliott, T. K. Broschat, J. Y. Uchida, and G. W. Simone (eds.). The American Phytopathological Society, St. Paul, MN. 607