Effect of the Growth Regulator Paclobutrazol on Growth of the Bacterial Pathogen Xylella fastidiosa

• Bacterial leaf scorch
• oxytetracycline
• paclobutrazol
• Xylella fastidiosa

Xylella fastidiosa (Wells et al. 1987) is a xylem-limited, insect-transmitted, fastidious plant pathogenic bacterium with a wide host range. It is currently recognized as indigenous to the Americas and causes disease in grape, citrus, shade trees, and other hosts. Pierce’s disease of grape, caused by the bacterium, was first described by Newton B. Pierce in 1892 (Pierce 1892) as an irregular marginal necrosis that resulted in the slow systematic death of the vine. Transmission of the bacterium by arthropods of the Homoptera was confirmed by Houston et al. in 1947. In the 1950s, marginal leaf scorching symptoms similar to Pierce’s disease were observed on American elms (Ulmus americana) in Washington, DC, U.S. (Wester and Jylkka, 1959) leading to a hypothesized relationship between the syndromes. The isolation of a xylem-limited bacterium confirmed to be the casual agent of Pierce’s disease (Davis et al. 1978) subsequently enabled scientists to implicate this bacterium as the cause of a variety of syndromes, including elm leaf scorch (Hearon et al. 1980), phony disease of peach (Hopkins and Mollenhauer 1973), and almond leaf scorch (Mircetich et al. 1976). Analysis of 25 isolates of the pathogen from 10 distinct hosts in 1987 allowed Wells et al. to describe and name the organism X. fastidiosa as a new genus of plant pathogenic bacteria represented by a single species. In 2000, X. fastidiosa became the first plant pathogen to have its entire genome sequenced (Simpson et al. 2000).

Xylella fastidiosa infection in shade trees is commonly referred to as bacterial leaf scorch (BLS) and the causal role of the bacterium was first demonstrated by Sherald et al. (1983) in sycamore (Platanus occidentalis). Xylella fastidiosa has a wide host range affecting over 100 host species in at least 30 families of monocotyledonous and dicotyledonous plants (Hopkins and Alderz 1988), including such important landscape trees as oaks (Quercus), elms (Ulmus), sycamores (Platanus), and mulberries (Morus) in urban or suburban areas of the mid-Atlantic and southeastern United States (Sherald 2001). Xylella fastidiosa has been confirmed to infect numerous tree hosts (Table 1) throughout eastern North America and California, U.S. Most hosts are asymptomatic and may serve as reservoirs of the pathogen from which vectors transmit the disease to susceptible hosts. Xylella fastidiosa is transmitted by xylem-feeding insects of the Cicadellinae (sharpshooters/leafhoppers) and Cercopidae (spittlebugs), which are active throughout the growing season and remain infectious indefinitely after bacterial acquisition.

Bacterial leaf scorch is a chronic late season leaf scorch that compromises the growth and aesthetic qualities of shade trees, although it is rarely lethal. Symptoms of the disease include an irregular marginal leaf scorch, early defoliation, delayed budbreak, loss of vigor, stunting, and dieback. Scorch symptoms result from the disruption of water transport attributable to bacterial aggregation in xylem vessel elements and are usually apparent by late summer with severity varying annually as a function of moisture availability (McElrone et al. 2001). Affected trees will appear less vigorous with a noticeable reduction in growth over the long term. The bacteria spread systemically through the xylem of the tree; symptoms typically begin on a single branch and progress to adjacent branches and ultimately the entire canopy over several years. The disease is commonly reported in urban and suburban areas and is less commonly seen in natural areas.

Treatment of BLS was first attempted using the antibiotic oxytetracycline with some success. However, treatment did not prove curative and symptom remission was highly variable (Kostka et al. 1985). Paclobutrazol (PBZ) is a diastereomeric triazole (Sugavanam 1984) that possesses properties of gibberellin (Radenmacher et al. 1987) and sterol biosynthesis inhibition (Burden et al. 1987). The compound reduces shoot growth, confers drought tolerance to plants (Frakulli and Voyiatzis 1999), and inhibits mycelial growth of a wide range of fungi (Jacobs and Berg, 2000). Paclobutrazol is considered a multiprotector as a result of its ability to ameliorate biotic and abiotic stresses in plants (Fletcher and Hofstra 1986). Application of PBZ to oak trees exhibiting BLS has been observed to alleviate symptoms (The Bartlett Tree Research Laboratories, pers. comm.); however, this remains to be validated through experimental studies. Currently, there is no evidence that PBZ directly inhibits growth or survival of X. fastidiosa. The purpose of this study was to determine whether PBZ inhibits growth of X. fastidiosa in vitro.

MATERIALS AND METHODS

Bacterial Isolates and Culture

Studies were conducted using X. fastidiosa isolates from elm (Ulmus americana) and grape (Vitis vinifera) grown on semisolid periwinkle wilt (PW) medium (Davis et al. 1981). Isolates had been held in storage at −70°C (−158°F). All strains were transferred to and maintained on buffered charcoal yeast extract (BCYE) semisolid medium (Feeley et al. 1979) and in PW broth at 28°C (82.4°F) and subcultured on a regular basis.

Standard Curve

A Spectronic Genesis 5 spectrophotometer (Spectronic Instruments, Inc., Rochester, NY) was used to measure the absorption at 600 nm of X. fastidiosa cells in PW broth cultures. Broth cultures with a range of cell densities were measured by spectrophotometer and subsequently diluted logarithmically to allow direct determination of bacterial colony-forming units, the number of separate bacterial colonies observed (CFUs) from the absorbance measures. Dilution series were executed by plating a 100 μL aliquot of each sample on BCYE followed by a 10-fold dilution; this was repeated until the 1 × 10¹⁰ dilution was achieved. Plates were incubated at 28°C (82.4°F) and the number of CFUs counted. Absorbance values were plotted as a function of the number of colonies and evaluated by regression analysis using statistical analysis software (SAS) to determine the best fit model for the calculation of CFUs from absorbance readings (Figure 1).

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Figure 1.

The relationship between absorbance of a periwinkle wilt broth sample measured by a spectrophotometer and amount of Xylella fastidiosa colony-forming units (CFUs) within the sample as determined by dilution plating.

Inoculum

Elm and grape isolates of X. fastidiosa were grown for 10 days on BCYE semisolid medium. Cells were scraped and suspended in PW broth. Cultures were incubated for 5 days at 28°C (82.4°F) and standardized to ≈1 × 10⁴ CFUs using a spectrophotometer.

Treatments

Oxytetracycline, 50 μg/mL⁻¹, was used as the treated control. A 50 μg/mL⁻¹ oxytetracycline solution was prepared by dissolving 5 mg of oxytetracycline in 10 mL (0.3 fl oz) PW broth. The broth was filter sterilized through a 0.22 μM Millipore membrane and added to 90 mL (2.7 fl oz) of the PW broth.

Three 10 mL (0.3 fl oz) stock solutions of technical-grade PBZ (Syngenta Crop Protection Inc., Greensboro, NC) were prepared in 95% ethanol at the concentration of 2000, 200, and 20 μg/mL⁻¹ and stored at room temperature. Aliquots of 100 μL from each PBZ stock were applied to 100 mL (3 fl oz) PW broth to make 200, 20, and 2 μg/mL⁻¹ PBZ solutions.

Growth Study

Growth studies were conducted using 1.5 mL (0.05 fl oz) sterile microcentrifuge tubes. Nine hundred microliter aliquots of each solution (control PW broth, 50 μg/mL⁻¹ oxytetracycline, 200 μg/mL⁻¹ PBZ, 20 μg/mL⁻¹ PBZ, 2 μg/mL⁻¹ PBZ) were added to 40 tubes each. Subsequently, all treatments received 100 μL of X. fastidiosa inoculum and were incubated for 10 days at 28°C (82.4°F). Uninoculated standards of each treatment served as references.

After incubation, three randomly selected microcentrifuge tubes from each respective treatment were removed each day for 10 days and absorbance determined by spectrophotometry. References of each respective media from the same media batch were used in duplicate to ensure consistency and accuracy. Additionally, a logarithmic series of dilutions in sterile water was prepared for a randomly selected sample from each treatment everyday to ensure purity of the cultures and accuracy of the spectrophotometer. Dilutions were plated each day for the first 5 days on BCYE and subsequently incubated for 10 days after which time bacterial colonies were counted.

RESULTS

Standard Curve

A simple linear regression equation [y = 0.0199x − 0.0939], where y is the number of CFUs and x is the absorption, was produced. The absorption depended on the CFUs in the broth culture. Therefore, inverse regression was used to calculate CFUs from absorbance measurements. Inverse regression was done by regressing absorbance values on CFUs, resulting in the equation [x = 50.25y + 4.7186].

Evaluation of the Effect of Paclobutrazol on X. fastidiosa by Spectrophotometery

Treatments of oxytetracycline at 50 μg/mL⁻¹ and PBZ at 200 μg/mL⁻¹ significantly reduced the growth of both the elm and grape isolates of X. fastidiosa as compared with the PW medium control or the PBZ treatments at 2 μg/mL⁻¹ and 20 μg/mL⁻¹ (Figure 2; Table 2). No significant growth difference was observed between the two X. fastidiosa isolates under any of the treatments (Figure 2; Table 2).

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Figure 2.

Growth curve of Xylella fastidiosa isolate elm (top) and grape (bottom) in periwinkle wilt (PW) broth supplemented with chemical treatments of either oxytetracycline (antibiotic) or paclobutrazol (PBZ) measured over 10 days with a spectrophotometer. Absorbance readings were converted to colony-forming units (CFUs) using the regression equation (Figure 1) determined for this study. Means and associated standard errors are plotted.

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Table 2.

Growth of Xylella fastidiosa isolates from elm and grape grown in periwinkle wilt broth supplemented with either oxytetracycline (antibiotic) or paclobutrazol (PBZ).^z

Evaluation of the Effect of Paclobutrazol on X. fastidiosa by Dilution Plating

Both oxytetracycline and PBZ at 200 μg/mL⁻¹ reduced the number of CFUs of X. fastidiosa isolates from elm (P < 0.01) and grape (P < 0.01) when compared with the control or low levels of PBZ (Table 3). However, in grape and elm tissue, PBZ was not as effective in delaying or reducing bacterial growth as oxytetracycline (Figure 3).

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Table 3.

Growth of Xylella fastidiosa isolates elm and grape growth in periwinkle wilt broth supplemented with either oxytetracycline (antibiotic) or paclobutrazol (PBZ).^z

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Figure 3.

Growth curve of Xylella fastidiosa isolate elm (left) and grape (right) in periwinkle wilt (PW) broth supplemented with chemical treatments of oxytetracycline (antibiotic) and paclobutrazol (PBZ) measured over 5 days. Colony-forming units (CFUs) were determined by dilution plating. Means and associated standard errors are plotted.

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