Blocking Phragmites australis reinvasion of restored ... - Springer Link

Wetlands Ecol Manage (2006) 14:539–547 DOI 10.1007/s11273-006-9006-6

ORIGINAL PAPER

Blocking Phragmites australis reinvasion of restored marshes using plants selected from wild populations and tissue culture Jiangbo Wang Æ Denise M. Seliskar Æ John L. Gallagher Æ Michael T. League

Received: 8 February 2005 / Accepted: 20 February 2006 / Published online: 28 July 2006

Springer Science+Business Media B.V. 2006 Abstract This study tested a vegetation strategy for controlling Phragmites australis invasion into brackish marshes as an alternative to the current technique of repeated herbicide sprays followed by burning. This strategy involves blocking P. australis by planting desired plants selected from wild populations and/or tissue culture regenerants at key points on the major routes of P. australis invasion. The planting of native species was conducted at three sites in a herbicide-treated P. australis marsh near Salem, NJ. Wild population selections of three upland marsh shrubs, Myrica cerifera, Baccharis halimifolia, and Iva frutescens, as well as two grass species, Spartina alterniflora and Spartina patens, and two rushes, Juncus gerardi and Juncus roemerianus, were planted according to their normal zonation positions. Tissue culture regenerated plants of the two grasses and two rushes, and the sedge species Scirpus robustus, were also planted. Plant growth at each site was monitored each year after planting for up to 3 years. Most plants of B. halimifolia, I. frutescens, J. roemerianus, and S. patens demonstrated a consistent vigorous growth at all three sites, whether or not the plants were collected from wild

J. Wang Æ D. M. Seliskar (&) Æ J. L. Gallagher Æ M. T. League Halophyte Biotechnology Center, College of Marine Studies, University of Delaware, 700 Pilottown Road, Lewes, DE 19958, USA e-mail: [email protected]

populations or were tissue culture regenerants. These multi-layered walls of plants demonstrated effectiveness in controlling the P. australis by restricting or inhibiting its spread. Upon screening 48 regenerated plants of S. patens at one of the three sites, we found that some regenerants showed enhanced characteristics for blocking P. australis, such as greater expansion and a high stem density. The availability of the tissue culture-regenerated plants of the native marsh species makes it possible to select lines from local genotypes that have desirable characteristics for wetland restoration projects, such as blocking P. australis reinvasion. Keywords Invasion Æ Phragmites australis Æ Wild populations Æ Tissue culture-regenerated plants Æ Wetland restoration

Introduction Phragmites australis (Cav.) Trin. ex. Steud. is native to American wetlands. Over the past century, a genotype of this species introduced from Europe has invaded many wetlands of the United States (Saltonstall 2002) and resulted in detrimental effects to the ecosystem. There are three major routes of the invasion: (a) from the upland to the marsh plain vegetatively by rhizomes or stolons, (b) from other marshes as pieces of vegetative material carried by the

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tide and stranded on ‘‘high spots’’ in the marsh where they can root and grow, or (c) from seeds that are transported by the wind or tidal water to ‘‘high spots’’ in an otherwise rather low marsh plain. Once established in the more benign habitats, P. australis can grow down into the lower, more stressful sites where it could not initially become established. For example, P. australis cannot germinate in the low anaerobic areas (Wijte and Gallagher 1996a, b), but it can grow into anaerobic areas from where it is well-established. Likewise, P. australis seedlings are more sensitive to sulfide than Spartina alterniflora seedlings (Seliskar et al. 2004), but if first established in an aerobic site, it can spread into areas of higher sulfide. Currently, the common way to control P. australis is to spray the plants with a glyphosate herbicide in the fall and, in some areas, burn the dead canes in the spring, for two consecutive years. Desirable plants usually recolonize the barren sites without human intervention, but very often P. australis reinvades within a few years. The solution is to repeat the spray and burn practices, which are followed by ‘‘down years’’ due to the loss in desired wetland functions for living aquatic resources. This treatment also has economic and environmental shortcomings such as labor costs and air pollution. A vegetation alternative to the cyclic spray and burn for P. australis control is to block P. australis by planting native marsh species at the key points on the major routes of P. australis invasion. Such a point can be a barren ‘‘high spot’’ that may be easily colonized by P. australis, or a spot between the upland and marsh plain from where P. australis invasion or reinvasion may start. Wild-type selections of the native species can be tested for their blocking potential. It is also applicable to use tissue culture regenerated plants. Tissue culture has been suggested to be an efficient tool for providing the large numbers of plants required in wetland creation and restoration (Kane 1996; Rogers 2003; Wang et al. 2004). Meanwhile, tissue culture is a process that usually produces somaclonal variation (Larkin and Scowcroft 1981; Phillips et al. 1994; Vazquez 2001). Numerous studies on improving crop cultivars by exploiting somaclonal variation have been reported (Winicov 1991; Oberthur et al. 1993; Racchi, et al. 1995; Bertin et al. 1997; Chintapalli et al. 1997). It is possible to select lines of regenerated plants with characteristics that make it superior to the wild-type

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for blocking P. australis more effectively. Characteristics, such as a dense shoot and root system, a thick upright shading canopy, and possibly the production of secondary metabolites that would form an allelopathic barrier against P. australis, are desirable in such a selected line. In this study, selected plantings of native species were conducted at three sites in a previously herbicide-treated P. australis marsh. Plants collected from wild populations of three upland marsh shrubs, Myrica cerifera L., B. halimifolia L., and I. frutescens L., as well as two grass species, S. alterniflora Loisel., S. patens (Aiton) Muhl, and two rushes Juncus gerardi Loisel., and Juncus roemerianus Scheele, were planted. Tissue culture-regenerated plants of the two grasses and two rushes, and a sedge species, Scirpus robustus Pursh, were also planted. The growth and spread of these plants was quantified. P. australis shoot number was compared within our test planting sites and adjacent control sites.

Methods Site planting Three sites in a P. australis-dominated wetland being restored to a pre-invasion species assemblage near Salem, New Jersey were selected for the experimental planting. These sites form the transition zone between the upland and the marsh plain and have approximately a 1 slope. At the time our study was initiated the marsh plain was dominated by S. alterniflora, while the upland/marsh fringe still contained P. australis. The dimensions (length · width) of the three sites were: 9 m · 7.8 m for Site 1; 7.5 m · 3.5 m for Site 2; and 17.1 m · 5.5 m for Site 3. There was approximately a 15 cm difference between the high and low points of each of the sites in elevation. The soil type of the sites is silty loam. All the sites were dominated by P. australis before being sprayed by herbicide in 1999. Site 1 was barren after removing the dead canes of P. australis before the planting in 2000; however there was live P. australis on the upland edge at this site. At Site 2, P. australis had come back before the planting in 2001. Site 3 was dominated again by P. australis before the planting in 2002. The above-ground P. australis at Sites 2 and 3 was cut prior to the planting.


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Both wild population and tissue culture-regenerated plants were prepared for the site planting. The wild population M. cerifera, I. frutescens, B. halimifolia were well-established juvenile plants dug from the upland fringe of Delaware local marshes, while those of S. patens, J. gerardi, and J. roemerianus were collected from mature stands in Delaware marshes. These donor sites are within 50 miles direct distance to the New Jersey experimental sites. For the S. alterniflora collected from wild populations, six plants originated from several Georgia marshes and two from a Virginia marsh. While the ecological setting of the Virginia S. alterniflora is similar to that of Delaware and New Jersey, the Georgia plants at their site of origin experience greater tidal amplitudes and a longer growing season. Interestingly, our work on S. alterniflora has demonstrated that in a common garden marsh in Lewes, Delaware, Georgia plants have done well for greater than 10 years (Seliskar et al. 2002). Based on this data, we expect that the Georgia S. alterniflora planted at the New Jersey site will do well in the long-term because of the close proximity of the Lewes and New Jersey sites. Regenerated plants were obtained by using tissue culture protocols developed for J. roemerianus and J. gerardi (Wang et al. 2005), S. robustus (Wang et al. 2004), S. alterniflora (Wang et al. 2003), and S. patens (Li et al. 1995). All the regenerated plants had been well-established in their pots in the greenhouse prior to planting in the field sites. Only wild population plants were planted at Site 1. Both wild and tissue culture-regenerated plants were

planted at Sites 2 and 3. All plants were planted at each site according to their natural zonation patterns. M. cerifera, I. frutescens, B. halimifolia, and J. gerardi were planted in the high zone, followed by S. patens and J. roemerianus in the middle zone, and S. robustus, and S. alterniflora in the low zone. Within each species at each site, the number of ramets per planting were similar. For plants collected from wild populations of each species, clumps of plants of similar sizes were transplanted. For the tissue culture-regenerated plants at each site, plants grown and well-established in the same size pots were transplanted. The origin of the plants, either from wild populations or tissue culture regenerants, as well as the numbers of the different species planted at each site, are described in Table 1. There was an approximate 100 cm distance between plants at Site 1, and about 60–70 cm at Site 2. At Site 3, there was an approximate 100 cm distance between plants for M. cerifera shrubs, and 60–70 cm for the other species. These distance differences were due to space constraints because the shapes of the three sites were different. Data collection Plant survivorship of each species at each site was recorded in the second and third years after planting (Table 1). The heights and circumferences of the surviving plants were recorded each year. Times of planting and data collection for each of the three sites are also listed in Table 1. For the plants that survived

Table 1 Plant survivorship at the three test sites Plant species

Site 1

Site 2

Site 3

P M1 M2 P M1 M2 P M1 M2 July, 2000 June, 2001 July, 2002 Oct., 2001 July, 2002 July, 2003 Aug., 2002 July, 2003 Aug., 2004 N S S N S S N S S M. cerifera I. frutescens B. halimifolia J. gerardi S. patens J. roemerianus S. robustus S. alterniflora

8 (W) 10 (W) 8 (W) 12 (W) 12 (W) 35 (W) – 8 (W)

25 100 100 100 100 100 – 100

37.5 100 100 100 100 100 – 100

18 (W) – 6 (W) – 10 (TC) – 4 (TC) –

83.3 – 100 – 100 – 100 –

38.9 – 100 – 100 – 100 –

33 (W) – 37 (W) 6 (TC) 48 (TC) 6 (TC) 6 (TC) 6 (TC)

78.8 – 92.3 100 100 100 100 100

72.7 – 92.3 66.7 95.8 100 66.7 83.3

P: Planting time. M1: Time of the first measurement. M2: Time of the second measurement. W: Plants collected from wild populations. TC: Tissue culture-regenerated plants. N: Number planted. S: % survivorship. ‘‘–’’ indicates that the particular species was not planted at that site

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in both of the measurement years, the mean height and circumference, as well as the percentage change from the first measurement to the second measurement, were calculated. With regard to the 46 S. patens regenerants at Site 3 that survived in both measurement years, the median, minimum, maximum, 25th percentile, and 75th percentile were determined for height, circumference, and percentage change. Estimated plant cover was also determined for the 46 plants using the Daubenmire cover scale (Mueller-Dombois and Ellenberg 1974) in 2004. Cover was determined by placing a measuring tape around the outermost shoots of a particular plant and then estimating percent cover within that circumference. Cover classes were as follows: 0–1, 1–5, 5–25, 25–50, 50–75, 75–90, 95–100%. A plant stand was considered to be 100% covered if the stems had reached the highest possible density and no soil surface was visible. The cover measurement provides a density estimation of the spreading plant. In July of 2003, the stems of P. australis were counted in the J. roemerianus zone and the Iva & Baccharis zone at Site 1, and in a control zone adjacent to this site. The control zone was an extension of Site 1 in the direction parallel to the upland line. Three quadrats of 200 cm · 75 cm were randomly selected from each of the three zones (J. roemerianus, Iva/Baccharis, and control zones) for the P. australis stem count. In August of 2004, P. australis stems were counted in the S. patens zone at Site 3, and in an adjacent, equal elevation control zone. The entire S. patens zone was divided into three 570 cm · 150 cm quadrats for the P. australis count. Likewise, P. australis stems were counted in three 200 cm · 150 cm quadrats in the control zone. The quadrats at Sites 1 and 3 were equidistant from

the invading P. australis stands within each site. Data on P. australis stem density were calculated and were analyzed using one-way ANOVA and the Tukey test.

Results All plants of the seven species planted at Site 1 were collected from wild populations and their survivorship is listed in Table 1. Except for M. cerifera, the other six species at Site 1 demonstrated 100% survivorship in both 2001 and 2002. M. cerifera at this site showed low survivorship in 2001 (25%), although one plant that had appeared dead produced a new shoot from the base in 2002 increasing survivorship to 37.5%. For all species at Site 1, the mean height and circumference increased from 2001 to 2002 (Table 2). Except for J. gerardi, the other species showed large circumference increases. The two shrubs, I. frutescens and B. halimifolia, also demonstrated relatively large height increases. By 2003, all the non-shrub species at Site 1 had grown together so clumps were indistinguishable and therefore were not measured, but it was evident that the plants grew vigorously during the year since the last measurement. Table 1 also lists the survivorship of the M. cerifera and B. halimifolia collected from wild populations and those of the tissue culture regenerants of S. patens and S. robustus at Site 2. The survivorship of M. cerifera decreased from 83.3% in 2002 to 38.9% in 2003, while that of B. halimifolia and S. patens were 100% for both years. There was a 100% survivorship of the S. robustus in 2002. In 2003, however, the spread of the S. robustus was indistinguishable from that of the wild plants growing

Table 2 Plant growth from 2001 to 2002 at Site #1 Plant species

Mean height in 2001 (cm)

Mean circumference in 2001 (cm)



Percentage change in height from 2001 to 2002 (mean±SE)

Percentage change in circumference from 2001 to 2002 (mean±SE)

M. cerifera I. frutescens B. halimifolia S. alterniflora J. gerardi S. patens J. roemerianus

77.5 121 100.8 78.9 47.1 107 100.1

115.5 159 129.9 182.8 103.1 96.8 75.7

111 190.4 183 103.1 48.3 118 126.3

127.5 264.1 194.5 308.4 120.8 179.4 142.9

88.9±68.8 58.5±5.6 90.5±19.6 32.3 ±5.3 4.3±5.8 10.8±4.5 27.5±3.3

9±1.8 70.7±17 71.3±38.7 72.5±19.5 21.1±12.2 90.1±20.1 103.1±8

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into the planting site from the surrounding marsh. M. cerifera plants exhibited poor growth from 2002 to 2003, demonstrated by a negative change in circumference and no change in height (Table 3). Meanwhile, the B. halimifolia and S. patens grew vigorously with large increases in both height and circumference (Table 3). At Site 3, M. cerifera and B. halimifolia were collected from wild populations, while the plants of the other five species were tissue culture regenerants, and their survivorship is listed in Table 1. The plants of M. cerifera at this site showed a better survivorship (78.8% in 2003 and 72.7% in 2004) than those at the other two sites. The plants of B. halimifolia maintained a 92.3% survivorship in 2003 and 2004. For the other five species, all plants survived in 2003. However, only J. roemerianus maintained a 100% survivorship in 2004. The 2004 survivorship of both J. gerardi and S. robustus was 66.7%, while that of S. patens and S. alterniflora was 95.8 and 83.3%, respectively. Some of the growth characteristics of the plants at Site 3 are listed in Table 4. Except for S. robustus and S. alterniflora, the plants of the other five species showed increased mean height and circumference. The height increase of B. halimifolia was relatively large. This species, along with M. cerifera and J. roemerianus, also showed large circumference increases. For S. robustus plants, a small increase in height and a decreased circumference were observed, while both the height and circumference of S. alterniflora decreased. Forty-six of 48 tissue culture regenerants of S. patens survived to 2004 at Site 3. Table 5 shows the median, minimum, maximum, 25th percentile and 75th percentile of circumferences of these plants in 2003 and 2004, and the percentage change in circumference from 2003 to 2004. Table 6 shows the number of plants that fell into each of the seven classes of the Daubenmire cover scale. No plant was found in the top 25% levels of all three categories

of circumference change, i.e. above 85.5 cm in 2003 circumferences, above 116.25 cm in 2004 circumference, and above 74.4% in percentage change in circumference. However, there were seven plants in the top 25% levels of 2003 and 2004 circumferences. Two of the seven plants, designated as R28 and R35, also had a cover over 75%. Regarding regrowth of P. australis into the planted sites, at Site 1, the P. australis stem densities were 8.6 stems per m2 in the Iva and Baccharis zone and 25.1 stems per m2 in the J. roemerianus zone, which were significantly lower than that of 149.1 stems per m2 in the control zone (a=0.05) (Fig. 1A). The P. australis stem density in the S. patens zone at Site 3 was 6.9 stems per m2, which was also significantly lower than that of 66.2 stems per m2 in the control zone (a=0.05) (Fig. 1B). Both control zones were dominated by P. australis at the time of stem counting.

Discussion Although located in the same zone of the same marsh, the three sites varied because the year of planting was different and the time following herbicide treatment varied. Site 1 was barren after the herbicide treatment of P. australis in the previous year. There are two major sources for the reinvasion of P. australis at this site. One is the P. australis at the upland edge. The other is the in-site live rhizomes that survived the herbicide and that still have the ability to produce new shoots. P. australis reinvasion occurred at Site 2 and Site 3 before the plantings in 2001 and 2002, respectively. The plant growth at each of the three sites was affected by different stages of in-site P. australis reinvasion. In addition, there was precipitation variation in southern New Jersey throughout the years of the study (New Jersey historical precipitation data, Rutgers University). In 2004, it


Mean height Mean Mean height Mean Percentage change Percentage change in 2002 (cm) circumference in 2003 (cm) circumference in height from 2002 in circumference in 2002 (cm) in 2003 (cm) to 2003 (mean±SE) from 2002 to 2003 (mean±SE)

M. cerifera 37.6 B. halimifolia 39.2 S. patens 34.2

51.8 56.6 26.7

32.6 138.4 112.2

42.6 134 91.7

0.4±15.8 252.2±11.3 240.9±15.3

2.9±28 136.8±37.3 250.8±23.4

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Percentage change in height from 2003 to 2004 (mean±SE)

Percentage change in circumference from 2003 to 2004 (mean±SE)

M. cerifera B. halimifolia S. patens J. gerardi J. roemerianus S. robustus S. alterniflora

108.2 55.6 69.1 30.3 79.2 90.8 94

60.1 34.9 63.4 39.3 42.7 179 69

108.2 96 85.7 32.8 112.5 103.3 86

131.2 107.9 87.4 60.5 136.8 41.8 52

8±4.1 73.4±5.6 25.7±2.9 9.7±9.2 42.4 ±5.4 15.1±8.9 9.4±5.8

165.6±25.3 233.2±25.2 46.4±8.9 53±33.5 232.6±42.4 78±6.2 15.7±31.9

Table 5 The median, minimum, maximum, 25th percentile and 75th percentile of circumferences of S. patens regenerants in 2003 and 2004, and the percentage change in circumference from 2003 to 2004

Median Minimum Maximum Percentile (25th) Percentile (75th)

Circumference in 2003 (cm)

Circumference in 2004 (cm)

Percentage change in circumference from 2003 to 2004

60.5 13 124 43.5 85.5

87.4 90.5 4 60 116.3

40.6 88 186 12.9 74.4

Table 6 The number of S. patens regenerated plants that fell into each of the seven classes of the Daubenmire cover scale Daubenmire classes of percent cover

0–1%

1–5%

5–25%

25–50%

50–75%

75–90%

95–100%

Number of plants

3

5

9

13

11

5

0

was observed that the S. alterniflora in the restored marsh was dramatically infiltrated by Amaranthus cannabinus, tidalmarsh amaranth, an annual weedy plant that is common in brackish and freshwater wetlands. This was probably caused by the above average precipitation in 2003, which resulted in low soil salinity. The precipitation variation or other environmental factors may also have affected the growth of the plants in the three experimental sites. For example, the above average precipitation in 2003 may have led to the negative growth of S. robustus and S. alterniflora regenerants at Site 3 from 2003 to 2004 (Table 4). Although there were site differences at the times of planting and precipitation variation throughout the years of study, most of the I. frutescens, B. halimifolia, J. roemerianus, and S. patens demonstrated a consistent vigorous growth at all three sites, whether or not the plants were collected from wild populations or were tissue culture regenerants. It appeared

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that P. australis spread was significantly inhibited by the Iva & Baccharis zone and J. roemerianus zone at Site 1, and by the S. patens zone at Site 3. I. frutescens and B. halimifolia individuals exhibited large canopies, which probably inhibited P. australis growth by shading. P. australis was inhibited in the S. patens and J. roemerianus zones where their dense shoot and root systems formed a physical barrier. The well-adapted species at these sites, especially I. frutescens, B. halimifolia, J. roemerianus, and S. patens, developed into multi-layered walls of plants that appeared to be effective in significantly blocking P. australis. Meanwhile, at each site, M. cerifera, J. gerardi, and S. robustus demonstrated poor survivorship or growth (Tables 1–4). These species are probably not good choices for blocking P. australis invasion. P. australis often out-competes S. alterniflora in brackish marshes, which had been the case for this New Jersey marsh. In our experimental sites, the


Number of Phragmits stems per m

2

200 180

A

2

b

160 140 120 100 80 60

a

40

a

20 0

Number of Phragmites stems per m

545

Zone 1

Zone 2

Zone 3

80

B

b

60

40

20

a

0

Zone 1

Zone 2

Fig. 1 (A). P. australis stem count in Site 1 in July, 2003. Zone 1: J. roemerianus zone; Zone 2: Iva and Baccharis zone; Zone 3: Control Zone. (B) P. australis stem count in Site 3 in August, 2004. Zone 1: S. patens zone; Zone 2: Control Zone. Bars represents mean ± SE. Bars marked by the same letter indicate that values are not significantly different from one another at the 5% level as determined by one-way ANOVA and Tukey test

S. alterniflora plants from Georgia and Virginia were taller and more robust (approximately 1 m in height) than the original on-site plants (approximately 0.5 m in height). Aerial biomass of S. alterniflora from Georgia and Virginia has been reported to be higher than that from Delaware and New Jersey (Reimold 1977). Seliskar et al. (2002) found that S. alterniflora from Massachusetts, Delaware, and Georgia maintained distinct morphologies, characteristic of morphologies at their sites of origin, even after 6 years of growth in a common garden followed by 5 years of growth in a created marsh in Delaware. Therefore, it is probable that the transplanted Georgia and Virginia plants planted in our New Jersey site have maintained the ability to produce a large biomass, and therefore may be more competitive against P. australis.

Currently, plant materials (such as clumps of plants and seeds) collected from the field or produced in a nursery are the primary sources for wetland creation or restoration projects. However, there are numerous concerns pointed out by the users of such plant material: (1) there is often a seasonal limitation in the field collection of the material and it is difficult to always have the plants ready for a particular window of planting; (2) field collection of plants can damage the marsh and is becoming restricted in some locations; (3) inappropriate plants may be introduced into the project sites if a clump of field collected plants is associated with the seed bank or plant material of other species; and (4) when vegetatively grown plants are used the genetic diversity is low. With the potential of alleviating such concerns and providing more benefits, tissue culture regenerated plants have been suggested as alternative sources for wetland creation and restoration (Kane 1996; Rogers 2003; Wang et al. 2004). Large numbers of plants can be propagated year round via tissue culture, making available a reliable and environmentally benign plant source during all seasonal windows of planting. Also, importantly, it is possible to take advantage of the somaclonal variation in the regenerated plants. Not only being able to produce regenerants with increased genetic variation, which could improve the long-term adaptability of these plants in an easily restored site, tissue culture has been exploited to select specific lines with characteristics desirable for sites requiring special plant features for rapid restoration (Seliskar 1998; Seliskar and Gallagher 2000). In a plant growth study in a simulated salt marsh, two of nine S. patens regenerants were found to spread vegetatively over a larger area and exhibit greater biomass and stem density (Wang et al. manuscript in press). These characteristics will be valuable for blocking P. australis. This same study also found genetic variation in the nine regenerants by using the random amplified polymorphic DNA (RAPD) technique. Genetic variation not only occurred among phenotypically different regenerants, but also among phenotypically similar ones. In the real world of wetland restoration, a concern may be that a selected line of tissue culture regenerant may become invasive. We believe this is a low risk for several reasons. In our study, only locally collected plants of native species from Delaware were tissue cultured. The existing invasive genotype

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of P. australis was introduced from another continent (Saltonstall 2002). There is no evidence that any of the species used in this study have become or will become invasive in their native region. For the native species studied here, no invasive behavior has been found in the marshes of the Atlantic coast. Basically, variation produced during the tissue culture process is similar to that which may occur in nature, but it can take place more rapidly in tissue culture because genomic stress in tissue culture enhances variation (Phillips et al. 1994). Considering the closeness of the New Jersey experimental site to Delaware, the non-invasive behavior, and the nature of somaclonal variation (occurring in nature), it is unlikely that tissue culture regenerants of native species in this study could develop into invasive strains. However, by employing adaptive management techniques, any signs of invasiveness can be addressed. All of the species used in our study can be controlled by herbicides. Development of high herbicide resistance via tissue culture is unlikely in a sensitive species without multiple in vitro exposures to selective pressure. In this study, it was found that seven out of 48 S. patens regenerants at Site 3 were in the top 25% levels of 2003 and 2004 circumferences. Two of the seven plants, R28 and R35, also had relatively high stem densities with covers over 75%. R28 and R35 are good candidates for blocking P. australis. The other S. patens regenerants that exhibited either large circumferences, or large percentage increases in circumference, or high stem densities, may also have potentials for the purpose of blocking P. australis. In a real world creation or restoration project where there is a need for blocking P. australis invasion, it is desirable to use as many different regenerants with desired blocking characteristics as possible for the sake of genetic diversity. In the field, the selected lines will reproduce vegetatively and therefore not lose their good morphological blocking characteristics. By considering both the ecological functions provided by the regenerants and the genetic diversity of plants for sustaining such functions, the chance of project success is increased; i.e. P. australis control is achieved while resiliency of the wetland plant community to other stresses is maintained. The implications of tissue culture regenerated plants for wetland creation and restoration have been discussed for Sporobolus virginicus (Seliskar 1998),

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Distichlis spicata (Seliskar and Gallagher 2000), S. alterniflora (Wang et al. 2003), S. robustus (Wang et al. 2004), and J. roemerianus and J. gerardi (Wang et al. 2005). In the present study where the latter four species were planted, only a small number of tissue culture regenerated plants of these four species were planted at Site 3. This limits the analysis of their potential for blocking P. australis invasion and the testing of more regenerants, as was done in the present study at Site 3 with S. patens, will be necessary in future research. In general, our strategy for blocking P. australis invasion is based on planting selections from wild populations and/or tissue culture regenerants if they have the desired characteristics, and growing a multilayered wall system of plants at the key points on the major routes of P. australis invasion. This strategy has demonstrated effectiveness in controlling P. australis, as shown by the P. australis stem counts in the planted and control zones. It will be desirable to test the strategy on a larger scale, with the aim of further developing an alternative herbicide-free and maintenance-free means of P. australis control. Acknowledgements Support for this research came from the Estuary Enhancement Program of the Public Service Enterprise Group, Salem, New Jersey and the University of Delaware Sea Grant College Program Project R/ME-24 under Grant No. NA96RG0029. The authors thank Colleen Butler, Rebecca Rush Clanton, Matthew Cloves, Joseph Klein, Cecelia Linder, Shawn Shotzberger, and Kayti Tigani for their help in the field, and especially Brenda Evans for her help with field site access, help in the field planting, and grant administration.

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