Multiplication and Translocation of Xanthomonas campestris pv. poae (JT-P482) in Annual Bluegrass (Poa annua L.)

Transcription

1 Multiplication and Translocation of Xanthomonas campestris pv. poae (JT-P482) in Annual Bluegrass (Poa annua L.) Seiko Imaizumi* and Takane Fujimori* Abstract: Growth of a Rif ampin-resistant strain (Rif-482) of Xanthomonas campestris pv. poae (JT-P482) in annual bluegrass monitored over several weeks indicates its potential as a tool for the study of interaction between X. campestris pv. poae (JT-P482, wild type) and annual bluegrass. After inoculation with 109 cfu/ml of bacterial suspension, populations of both Rif-482 and JT-P482 in plant sections increased during the period from 3 days (1.0x108 cfu/g FW) to 3 weeks after inoculation, when they reached a maximum (1.5x1010 cfu/g FW). Subsequently, Rif -482 decreased to 5.4x108 cfu/g FW 9 weeks after treatment (WAT). Efficacy (=% control) increased up to 4 WAT (86% control and 88% control of plants inoculated with Rif -482 and JT-P482, respectively) with plants showing the blighted and dried symptoms characteristic of heavy wilting. These results showed that the multiplication rate and efficacy of Rif-482 in plants matched those of JT -P482. The time lag between the peak of bacterial population (>1010 cfu/g FW) and the peak of disease development (>80% control) was between one and two weeks. Tests of various initial inoculum concentrations of both isolates from 103 to 1010 cfu/ml showed that the two followed a similar pattern of cell population increase and efficacy 3 WAT. The multiplication pattern of Rif-482 * Plant Protection Research Laboratory, Japan Tobacco Inc. 6-2 Umegaoka, Aoba-ku, Yokohama, 227 Japan (Accepted December 6, 1996) in the plant during the vegetative to early reproductive phases of plant growth was traced in different plant parts over time. Our findings show that Rif-482 bacteria inoculated to a leaf translocate systemically from the inoculation site through the stem to the root, then to all plant-parts. They also show a correlation between the development of disease and a decrease in the water content of plants, which appears to be the mechanism by which X campestris pv. poae injures annual bluegrass. Key words; bioherbicide; Xanthomonas campestris pv. poae (JT-P482); Rifampinresistant Xanthomonas campestris pv. poae; annual bluegrass (Poa annua L.); bacterial multiplication Annual bluegrass and translocation. Introduction is one of the most persistent weeds in turf, particularly in golf courses. The tufted, bunched growth pattern and prolific seed production of annual bluegrass interferes with uniformity of growth and color in cultivated turf2). Due to fear of damage to desirable turf grasses such as bentgrass (Agrostis palustris Huds.) and Kentucky bluegrass (Poa pratensis L.), chemical herbicides are not generally used to control it; as a result, annual bluegrass has been able to proliferate in managed turf grasses. A highly selective herbicide for its eradication has been sought by course superintendents for a considerable time4). Xanthomonas

2 116 J. Weed Sci. Tech. Vol. 42 (1997) campestris pv. poae which causes heavy bacterial wilt on annual bluegrass was identified in the U.S. as a possible selective biocontrol agent for this weed10,12) Also, X. campestris pv. poae (isolate JT-P482, wild type) was isolated in Japan in 19935) and has been developed as a bacterial bioherbicide, as previously reported6,7). Using histochemical techniques, bacterial movement of X campestris pv. poae within the xylem vessels was characterized in juvenile annual bluegrass plants1). It has been speculated that this pathogen invades the xylem and produces disease primarily by interfering with the movement of water. This report details our attempts to obtain a better understanding of the mechanism by which X campestris pv. poae (isolate JT-P482) injures annual bluegrass through an investigation of bacterial multiplication and translocation in adult plants. Prior to these experiments, a rifampin-resistant mutant of JT-P482 (=Rif -482) was selected and tested as a tool for the measurement of bacterial cell populations in plants. The incorporation of antibiotic (e.g., rifampin or streptomycin) resistance into plant pathogenic bacteria allows isolation of the bacteria from inoculated plant tissue plated on antibiotic-containing media without interference from contamination by other microorganisms. Thus antibiotic resistance has occasionally been used for studying the population dynamics of bacteria and in ecological studies14). Materials and Methods Plant material The annual bluegrass used in all experiments was grown from seed in 5cmx5cmx 5cm deep plastic pots filled with sand. Approximately 25mg of seeds (supplied by Utsunomiya University) were sown closely together, in the center of each pot. These pots were placed in a greenhouse (20C/15C, day/ night temperature) for 4 weeks by which time approximately 40 seedlings possessing 4-5 leaves each had grown in each pot. These plants were used for a sampling technique comparison and for the Rif-482 verification tests: a comparison of the multiplication rates and efficacy of JT-P482 and Rif For monitoring bacterial multiplication and translocation of Rif -482 in plants, some of these 4 week-old annual bluegrass seedlings were transplanted individually, one plant per pot, into 9cm diameter, 7.5cm deep plastic pots filled with sand. About one month after transplantation these plants had approximately 12 stems each, were in the vegetative stage of their life cycles, and were suitable for use in tracing the movement and population of the bacteria in plants. In all experiments, the sand in the pots was saturated with nutrient solution (N:P:K= 10:5:5) once a week throughout the testing period. Daily watering was carried out during all test periods. Rifampin-resistant mutant of Xanthomonas campestris pv. poae (JT- P482) To select a rifampin-resistant JT-P482 (Rif-482), 1ml aliquots of 24hr-old bacterial cultures (about 109 cfu/ml) were inoculated into 100ml of YNB medium (yeast extract 5g/liter and nutrient broth 8g/liter) containing rifampin (30pg/ml). After incubation overnight, rifampin-resistant colonies were spread on YN agar (yeast extract 5g/liter and nutrient agar 23g/liter) containing rifampin (3Opg/ml), and suitable colonies were selected. For long-term storage, Rif-482 was sub-cultured for 30h in YNB medium containing rifampin (3Opg/ml), at 28C, 200 rpm, then the broth was centrifuged for 15 min at 5000rpm (=2000g). Twenty per cent

3 Imaizumi and Fujimori: Multiplication and Translocation of Xanthomonas campestris pv. poae in Annual Bluegrass. 117 glycerol was added to the pellet, which was stored in a cryovial at -80C. The final concentration of colony forming units (= cfu/ml) was approximately 3X1011. Inocula preparation Cryovials containing Rif-482 and JT-P482 were thawed separately with sterile distilled water, and inoculum concentration was quantified by measuring optical density at 575nm to make final concentrations, as previously reported7). Accurate determination of the number of viable cells in a bacterial suspension was made on YN agar containing cycloheximide (30pg/ml) for JT- P482 detection, and YN agar containing rifampin and cycloheximide (30pg/ml in by the dilu- each case) for Rif-482 detection, tion-plate counting method. Sampling technique comparison Annual bluegrass seedlings clip-inoculated7), with 109 cfu/ml of Rif -482 were randomly sampled 3 weeks after inoculation. Eight leaves (approximately 0.1g) were arranged side by side with their apices alternately facing up then down, and the leaves were then divided equally. Half of the resulting leaf tissue was used for the fine-cutting technique, and the other half for the grinding technique. In the fine-cutting technique, samples were cut transversely to widths of about 0.5mm with sterile scissors, then put into 1ml of distilled water in a 2ml Eppendorf microtest tube. Test tube contents were mixed vigorously three times using a vortex mixer under room temperature conditions. In the grinding technique, samples were ground in a sterile mortar with a sterile pestle, then samples were moved to a 2ml Eppendorf tube and mixed in the same manner as the finecutting technique. Tenfold serial dilutions of the sample suspensions were made as described earlier. Comparison between Rif-482 and JT-P482: multiplication and efficacy Pots of annual bluegrass, each pot containing approximately 40 seedlings, were clipinoculated, half with 109 cfu/ml of Rif-482 and half with 109 cfu/ml of JT-P482, using sterile scissors. The clipping height was 2cm above the surface of the sand. Plants were then placed in the greenhouse (25C/20C). About g of test sample material was randomly selected from inoculated plants, and bacterial populations in plants were counted 3 days, 1, 2, 3, 4, 6, and 9 weeks after inoculation. Data are averages from 2-4 plants with six replications. In total, seven experiments with six replications were carried out for each isolate. To measure efficacy, a similar number of plants were inoculated as above with either Rif-482 or JT -P482 and a check treatment was prepared, cut and treated with sterile distilled water only. Efficacy was determined based on the measurement of fresh weight loss (=% control) of the entire top growth per pot compared with the check treatment8). Since this pathogen causes heavy wilting and death in annual bluegrass, the change in plants can be ascertained simply through fresh weight loss. Measurement took place at the same time intervals as stated above. Seven experiments with 6 replications were carried out for each isolate. Comparison of multiplication and efficacy of Rif-482 and JT-P482 at different inoculum concentrations Different bacterial suspensions of between 103 and 1010 cfu/ml of Rif-482 and JT-P482 were inoculated to annual bluegrass as described above. Each suspension was inoculated to six replicate pots in the test of multipli-

4 118 J. Weed Sci. Tech. Vol. 42 (1997) cation rates. In the efficacy tests, in addition to the pots inoculated with the different suspensions, a check control clipped only with sterile distilled water was also replicated six times. Bacterial populations in plants were counted and fresh weight was measured 3 weeks after inoculation. Bacterial multiplication and translocation in annual bluegrass Single annual bluegrass plants were inoculated with 109 cfu/ml of Rif-482 suspension to one fully expanded leaf tip per plant, using the clipping method. While inoculation was being carried out, an electric vacuum cleaner was employed to collect any excess inoculum and to ensure that it did not adhere to any other parts of the plant being inoculated. After inoculation, plants were placed in the greenhouse (20C/15C). Bacterial populations in different parts of the plant were monitored immediately (approximately 30 mm), and 1 and 3 days, 1, 2, 3, and 6 weeks after inoculation. The following parts of the plant were sampled as shown in Fig. 3: (1) a 2cm apical section of the inoculated leaf, (2) a 2cm section of the base of the stem bearing the inoculated leaf, measured from the soil surface up, (3) a 2cm section of root measured from 1cm below the soil surface down (the section was rinsed in water and excess water was removed using a paper towel), (4) a 2cm apical section of the leaf above the inoculated leaf, attached to the same stem, (5) a 2cm section of a tiller attached to the same stem as the inoculated leaf, measured from 1cm from the stem up, (6) a 2cm section of another stem connected to the stem bearing the inoculated leaf, measured from 1cm above the soil surface up, (7) a 2cm section of a stem newly emerged at the base of the plant about 10 days after inoculation, measured from 1cm above the soil surface up. Six pots each containing a single annual bluegrass plant, were sampled to ascertain bacterial population at each measurement time. For measurement of efficacy, samples were taken from 10 pots of inoculated plants and 10 pots of check control plants which had been clipped with sterile distilled water, 1, 2, 3 and 6 weeks after inoculation. Results and Discussion Sampling technique comparison To obtain a more convenient and timeefficient method for the measurement of bacterial populations in plants, our original fine-cutting technique was compared with the regular technique of grinding samples11). The time elapsing between the division of the leaf samples for each technique and the placing of those samples in the Eppendorf micro test tubes was 2 minutes in the fine-cutting technique and 5 minutes in the grinding tech- Table 1 Comparison of two sampling techniques, fine-cutting (A) and grinding (B), for recovery of rifampin-resistant Xanthomonas campestris pv. poae, Rif-482, in annual bluegrass.* *Leaves of annual bluegrass inoculated with 109 cfu/ml of Rif-482 were used. **Values are averages of six replications, + indicates the standard error. ***Samples were cut transversely to a width of about 0.5mm, then suspended in distilled water. ****Samples were ground in a sterile mortar with a sterile pestle, then suspended in distilled water.

5 Imaizumi and Fujimori: Multiplication and Translocation of Xanthomonas campestris pv. poae in Annual Bluegrass. 119 nique. The latter method is also more time consuming due to the necessity of sterilizing equipment. Thus the fine-cutting technique is particularly useful in dealing with large numbers of samples. Both techniques produced similar results when used to test for Rif-482, inoculated at rates of 109 cfu/ml (Table 1). After confirmation of this, the fine-cutting technique was used in all experiments to investigate bacterial populations in annual bluegrass plants. Comparison between Rif-482 and JT-P482: multiplication and efficacy Multiplication rates of Rif-482 in annual bluegrass were identical to those of JT-P482 (wild type) until 6 weeks after treatment (WAT) (Fig. 1). Populations of Rif-482 and JT-P482 in plant sections increased during the period from 3 days ( x108 cfu/g FW in the case of Rif-482, x108 cfu/ g FW in the case of JT-P482) to 3 weeks after inoculation, when they reached a maximum ( X1010 cfu/g FW of Rif-482, x1010 cfu/g FW of JT-P482). Then populations of Rif-482 decreased at 9 WAT ( x108 cfu/g FW). JT-P482 also decreased but was only detectable up to 6 WAT. Recovery of JT-P482 at 9 WAT was impeded by colonies of saprophytes which prevented the detection of these colonies on YN agar. Efficacy (=% control) shown in Fig, 1 was also quite similar until 9 WAT. In treatments with the two isolates, % control rates increased up to 4 WAT (86% control and 88% control of plants inoculated with Rif-482 and JT-P482, respectively). At this point, all inoculated plants showed the blighted and desiccated symptoms characteristic of heavy wilting. Thus, the bacterial multiplication, efficacy (=% control) and disease development of Rif-482 were demonstrated to be the Fig. 1 Multiplication rate of rifampin-resistant Xanthomonas campestris pv. poae, Rif-482, and wild type X. campestris pv. poae, JT -P482 in annual bluegrass plants (right hand scale) and the efficacy of both isolates in controlling annual bluegrass (left hand scale). The broken line indicates JT-P482. The unbroken line indicates Rif-482. The stages of disease development are noted. Four-week old plants were inoculated with 109 cfu/ml of bacterial suspensions of Rif -482 or JT-P482 under 25C/20C temperature conditions. Data are averages from 2-4 plants with six replications. Indications representing JT-P482 results have been placed slightly to the right of those representing Rif -482 in order that the error bar be visible.

6 120 J. Weed Sci. Tech. Vol. 42 (1997) same as those of JT-P482. The time lag between the peak of bacterial population (> 1010 cfu/g FW) and the peak of disease develoment (>80% control) was one to two weeks under these experimental conditions. After all treated plants had apparently died, 9 WAT, over 108 cfu/g FW of Rif-482 survived in plant tissue. Comparison of multiplication and efficacy of Rif-482 and JT-P482 at different inoculum concentrations Tests of various initial inoculum concentrations, from 103 to 1010 cfu/ml, showed similar rates of increase in bacterial populations in both Rif-482 and JT-P482 treated plants 3 WAT (Fig. 2). Initial inoculum concentrations of 103 to 107 cfu/ml in both isolates resulted in bacterial populations increasing gradually to x106 and x109 cfu/g FW of plants, respectively. High initial inoculum concentrations of 108 to 1010 cfu/ml also resulted in increased populations ( x1010 cfu/g FW). Results of treatment with JT-P482 and Rif-482 showed similar efficacy rates throughout the experiments. Initial inoculum concentrations of 1010 cfu/ml produced plant death 3 WAT. At inoculum concentrations of between 104 and 108cfu/ml, in neither isolate was there any correlation found between population size and efficacy, because higher populations were counted by simply averaging the cell numbers found in apparently healthy plants (c.f. 105cfu/g FW) and in wilting plants (c.f. 109 cfu/g FW). Rif-482 multiplication and translocation in annual bluegrass Systemic infection of annual bluegrass caused by Rif-482 was assessed by monitoring bacterial populations in various parts of the plant after inoculation of a single leaf Fig. 2 Multiplication rates of rifampin-resistant Xanthomonas campestris pv. poae, Rif-482, and wild type X campestris pv. poae, JT -P482 in annual bluegrass plants at various concentrations of inoculum (right hand scale), and the efficacy of both isolates in controlling annual bluegrass (left hand scale), at 3 WAT. The broken line indicates JT-P482. The unbroken line indicates Rif The stages of disease development are noted. Four-week old plants were inoculated with cfu/ml of bacterial suspensions of Rif-482 or JT-P482, under 25C/20C temperature conditions. Data are averages of 2-4 plants with six replications. Indications representing JT-P482 results have been placed slightly to the right of those representing Rif-482 in order that the error bar be visible.

7 Imaizumi and Fujimori: Multiplication and Translocation of Xanthomonas campestris pv. poae in Annual Bluegrass. 121 Fig. 3 Illustration of plant parts tested in assessment of translocation. (1) 2cm apical section of the inoculated leaf, (2) 2cm section of the base of the stem bearing the inoculated leaf, measured from the soil surface up, (3) 2cm section of root measured from lcm below the soil surface down, (4) 2cm apical section of the leaf above the inoculated leaf, attached to the same stem, (5) 2cm section of a tiller attached to the same stem as the inoculated leaf, measured from lcm from the stem up, (6) 2cm section of another stem connected to the stem bearing the inoculated leaf, measured from lcm above the soil surface up, (7) 2cm section of a stem newly emerged at the base of the plant 10 days after inoculation, measured from 1cm above the soil surface up. (Fig. 3). Inoculated Rif-482 moved from the inoculated leaf to the stem within approximately 30 min after inoculation, ( X 105 cfu/g FW at the site of inoculation and X102 cfu/g FW at the basal stem). Three days after inoculation, bacteria at the inoculation site increased to X107 cfu/g FW, and bacterial movement through the stem ( X105 cfu/g FW) to the root ( X104 cfu/g FW) was monitored. As shown in Fig. 4-A, Rif-482 spread to all plant parts systemically during the period from I to 3 weeks after inoculation, by which point the plant had wilted and was blighted. Efficacy data in Fig. l shows greater effectiveness and also faster rates of symptom development when compared with data from the multiplication test (Fig. 4-B). These differences were due to the different stages of plant growth (seedlings vs. mature plants), temperature conditions (25C/20C vs. 20C/ 15C) and cutting sites (many vs. one leaf). Commercially acceptable control rates of> 80% were achieved at 3 WAT in seedlings (Fig. 1) and at 6 WAT in adult plants (Fig. 4 -B). It is generally believed that bacterial growth ceases at the time of plant death, however, in the dead plants sampled in these experiments approximately X109 cfu/g FW of Rif-482 had survived in inoculated leaves, basal stems and roots 6 weeks after inoculation (Fig. 4-A). At this point, most plant parts could not be differentiated due to severe desiccation and the only data that could be measured came from the stem bearing the inoculated leaf and the root. A major vascular wilt pathogen causing bacterial leaf blight of rice, X. oryzae pv. oryzae, colonizes xylem vessels and produces extracellular polysaccharide3,13) This seems to protect the bacterial cells under dry or unfavorable conditions9). If a microorganism is to be used as a bioherbicide, it is important to trace how long it persists in the environment subsequent to use. An investigation into the persistence of X. campestris pv. poae (JT -P482) is currently underway. In addition to this it is important to know how the bacteria works within the plant. In

8 122 J. Weed Sci. Tech. Vol. 42 (1997) juvenile annual bluegrass, after entering the plant through a wound, this pathogen multiplies and moves from inoculated leaves to roots via the xylem1). The xylem is filled with bacteria, and then plants begin to wilt. It is supposed that this pathogen invades the xylem of stems and roots and produces disease primarily by interfering with the upward movement of water through the xylem. Our efficacy data were obtained simply by measuring of fresh weight loss, which reflected the decrease in water content caused by the development of disease. Our findings, (a) that bacteria inoculated to a leaf move systemically from the inoculation site through the stem to the root, then to all plant -parts, and (b) that the progression of disease development is correlated to a decrease in water content (= fresh weight loss), support this conclusion as to the mechanism by which X campestris pv. poae injures annual bluegrass. Fig. 4 Multiplication and translocation of rifampin -resistant Xanthomonas campestris pv. poae. (Rif-482) in various plant parts (A), and the efficacy of Rif-482 in controlling annual bluegrass (B), under 20C/15C temperature conditions. The stages of disease development are noted. Annual bluegrass plants, in the vegetative phase of growth, were inoculated with 109 cfu/ml of Rif-482 by clip-inoculation of one leaf tip. Data are averages, from 2cm sections of plant parts, of six replications for the (A) experiment, and ten replications for the (B) experiment. Numbers represent plant parts detailed in Fig. 3. Acknowledgement: The authors wish to thank Dr. M. Konnai of Utsunomiya University for the provision of the annual bluegrass seeds used in these experiments. We are grateful to Ms. R. Houkabe for her technical assistance and to Mr. Y. Nishino for his production of Rif-482. We also wish to acknowledge the assistance of Ms. Ann Mackie in the revision of this manuscript. References 1) Baragona, J. S Histopathology of juvenile Poa annua L. infected with Xanthomonas campestris. M. S. thesis. Louisiana Tech. Univ. pp ) Beard, J. B., P. E. Rieke, A. J. Turgeon and J. M. Vargas Jr Annual bluegrass L.), description, adaptation, (Poa annua culture and control. Res. Rep Michigan State Univ. Agric. Exp. Stan. pp ) Barton-Willis, P. A., P. D. Roberts, A. Guo and

9 Imaizumi and Fujimori: Multiplication and Translocation of Xanthomonas campestris pv. poae in Annual Bluegrass. 123 J. E. Leach Growth dynamics of Xanthomonas campestris pv. oryzae in leaves of rice cultivars. Phytopathol. 79, ) Branham, B Dealing with Poa annua. Golf Course Management. Sep ) Imaizumi, S., R. Houkabe, T. Nishino, Y. Omigawa and M. Yamada bluegrass with bioherbicide Control of annual (1) Efficacy testing on weed control of annual bluegrass with bacterium, Xanthomonas campestris. Weed Res., Japan 39, Suppl. I Abstr. (in Japanese). 6) Imaizumi, S., T. Nishino, M. Yamada and T. Fujimori Bioherbicidal control of annual bluegrass (Poa annua L.). Proceedings, of the 15th APWSS I (B) 7) Imaizumi, S., T. Nishino, K. Miyabe, T. Fujimori and M. Yamada Biological control of annual bluegrass (Poa annual.) with a Japanese isolate of Xanthomonas campestris pv. poae (JT-P482). Biological Control 8, ) Kalmowitz, K. E., T. J. Monaco, D. F. Ritchie and P. S. Zorner Xanthomonas annual bluegrass by temperature control of (Poa annua L.) is influenced and inoculum concentration. Proceedings of Southern Weed Science Society Abstr. 45, ) Ou, S. H Rice diseases. Kew, England Commonwealth Mycological Institute, Wallingford, U. K. pp ) Roberts, D. L., J. M. Vargas Jr. and R. Detweiler Occurrence of bacterial wilt on Poa annua and other turf grasses. Phytopathol. Abstr. 75, ) Robinson, J. N. and J. A. Callow Multiplication and spread of pathovars of Xanthomonas campestris in host and non-host plants. Plant pathol. 35, ) Savage, S. D Development of a strain of Xanthomonas campestris as a bacterial biocontrol agent for annual bluegrass (Poa annua) in amenity turf. Weed Sci. Soc. Am. Abstr. 31, ) Vidhyasekaran, P., M. E. Alvenda and T. W. Mew Physiological changes in rice seedlings induced by extracellular polysaccharide produced by Xanthomonas campestris pv. oryzae. Physiol. and Mol. Plant Pathol. 35, ) Weller, D. M. and A. W. Saettler Rifampin -resistant Xanthomonas phaseoli var. fuscans and Xanthomonas phaseoli: Tools for field study of bean blight bacteria. Phytopathol. 68,

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