Plant and Soil 266: 47–56, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
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Microbial communities of Solanum tuberosum and magainin-producing transgenic lines M. O’Callaghan1,4 , E. M. Gerard1 , N. W. Waipara2 , S. D. Young1, T. R. Glare1 , P. J. Barrell3 & A. J. Conner3 1 AgResearch,
PO Box 60, Lincoln, Canterbury, New Zealand, 2 Landcare Research, Mt Albert Research Centre, Private Bag 92170, Auckland, New Zealand and 3 New Zealand Institute for Crop and Food Research, Private Bag 4704, Christchurch, New Zealand. 4 Corresponding author∗
Received 13 March 2003. Accepted in revised form 10 October 2003
Key words: bacteria, fungi, magainin, potato, phyllosphere, rhizosphere, Solanum tuberosum, transgenic plants
Abstract Antimicrobial peptide magainin II, isolated from the skin of the African clawed toad, has shown activity in vitro against a range of micro-organisms. Transgenic potato lines expressing a synthetic magainin gene show improved resistance to the bacterial plant pathogen, Erwinia carotovora. Culturable bacterial and fungal communities associated with magainin-producing potato plants were compared with those communities from the non-transgenic parental control and with another potato cultivar. Total numbers of aerobic bacteria recovered from the leaves of the magainin-producing line, its non-transgenic parent line and an unrelated cultivar did not differ significantly. There were no detectable differences in the numbers of Gram-positive and Gram-negative bacteria, pseudomonad populations or fungi recovered from foliage from the three plant lines. Bacterial populations recovered from the roots of a magainin-expressing plant line did not differ significantly from populations recovered from the unmodified parental line. Tubers from the magainin-expressing transgenic potatoes, however, had significantly lower total numbers of bacteria than tubers produced by unmodified plants. In vitro testing of rhizosphere isolates against magainin analogues found that bacterial isolates varied in their susceptibility to the peptides. There were no significant differences in the total numbers of fungi and yeasts recovered from the various plant lines, with one exception: higher numbers of fungi were recovered from roots of magainin-expressing plants than the unmodified control plants.
Introduction Transgenic plants have been the focus of many risk assessment studies but effects of transgenic plants on key groups of micro-organisms have only recently been studied (Kowalchuk et al., 2003). Genetic modification of plants may lead to changes in the microbial flora beneficial or deleterious to plants which could alter the quality of agricultural soil (Heuer and Smalla, 1999). Several studies on impact of transgenic plants on micro-organisms associated with plants have been conducted in recent years: Lottman et al. (1999) and Heuer et al. (2002) examined the impact of T4lysozyme-producing potatoes on plant associated bac∗ E-mail:
[email protected]
teria, and Siciliano and Germida (1999) and Dunfield and Germida (2001) studied bacteria associated with the roots of herbicide resistant oilseed rape. Potatoes have been genetically modified for resistance to bacterial diseases by the incorporation of genes encoding various peptides and enzymes (Düring, 1996). For example resistance to the soft rot pathogen, Erwinia carotovora, has been developed by transgenic expression of T4-lysozyme (Düring et al., 1993), glucose oxidase (Wu et al., 1995) or tachyplesin I (Allefs et al., 1996) in potatoes. The transgenic potato plants investigated in this study were modified to express a synthetic magainin II gene, with the aim of protecting the plants from E. carotovora (Barrell, 2001). Magainin II is an antimicrobial pep-
48 tide isolated from the skin of the African clawed toad (Xenopus laevis), which has shown activity in vitro against a range of micro-organisms, including clinical isolates of some species of Gram-negative and Grampositive bacteria (Giacometti et al., 1998) and bacterial plant pathogenic species including Pseudomonas spp., Erwinia spp. and Clavibacter sp. (Alan and Earle, 2002). Synthetic magainin peptides have previously been shown to be active against yeast species Saccharomyces cerevisiae (Zasloff et al., 1988), and other plant-colonising and pathogenic fungi, including Penicillium, Alternaria, and Phytophthora spp. (Alan and Earle, 2002; Jacobi et al., 2000; Kristyanne et al., 1997). Transgenic tobacco and banana plants, modified to produce a magainin analogue MSI-99 showed enhanced resistance to a range of fungal pathogens, including Sclerotinia, Alternaria, Fusarium and Botrytis spp. (Chakrabarti et al., 2003). Unlike many other transgenic plants currently being developed, modification of potatoes to produce magainin II is being targeted at control of bacteria in the soil. Bioassays of tubers produced by transgenic plants against Erwinia carotovora showed less damaged tissue than unmodified control tubers (Barrell, 2001), indicating that sufficient magainin II was produced in the tubers to control Erwinia infection. Magainin II is secreted into the intercellular spaces in the plants and has the potential to change the structure of the microbial community associated with the leaves, roots and tubers. The objective of the present study was to develop methodologies and sampling strategies that would allow meaningful environmental impact assessment of transgenic potatoes in subsequent field trials. As little was known about potential impacts of magainin II on environmental micro-organisms, a preliminary characterisation of the bacterial and fungal communities associated with potato plants was carried out, with the aim of identifying particular groups of microorganisms that may warrant more detailed study in subsequent field trials. In this study, both epiphytic and endophytic fungal communities were also enumerated by viable plating and were identified to determine the diversity of fungal species. Materials and methods Plant material and field trial Transgenic and non-transgenic potato plants were sampled from the field trial in the 2001/2002 growing season at the New Zealand Institute for Crop
and Food Research, Lincoln, Canterbury, New Zealand (Environmental Risk Management Authority approval GMF98007). The potato lines D1, D2 and D3 were derived from Solanum tuberosum cv. Iwa using Agrobacterium-mediated transformation with a binary vector containing a kanamycin-resistant selectable marker gene and a synthetic magainin II gene under transcriptional control of a 35S promoter and a translational fusion to a signal sequence to direct export of the magainin II peptide into the intercellular space (Barrell, 2001). The magainin II gene included a translational fusion to the signal sequence from the tobacco PRS gene which directs export of proteins into the intercellular space after translation (Sijmons et al., 1990). All three transgenic lines were confirmed as producing correctly processed magainin II by western analysis which indicates successful transport out of potato cells (Barrell, 2001). Non-transgenic lines selected and sampled were parental cultivar Iwa, plus another potato cultivar Karaka. The trial was planted in December 2001 using tissue culture transplants as previously described (Conner et al., 1994), with final harvesting of tubers in May 2002. The nature of the field design was limited by regulatory restrictions imposed upon the trial approval. Each plot contained a row of 10 replicate plants 30 cm apart, with 75 cm between rows. Sampling of potato plants Foliage samples, consisting of the youngest fully expanded leaves, were collected 11 weeks after planting. Two replicate plots of Karaka, Iwa and D1 and single plots of lines D2 and D3 were sampled. To determine the optimum sampling strategy for use in subsequent studies, the variability in estimates of microbial populations resulting from sampling single plants in comparison with collection of composite samples (where plant material from three plants was pooled) was examined. Leaves from four single plants and two composite samples were collected from each plot. Roots and tubers were sampled at the time of harvest (senescent plants). Two plots of Iwa and D1 were sampled. From each plot, root and tuber samples were collected from four single plants and, for the two composite samples per plot, roots and tubers from three plants were collected into sterile bags, and treated as one sample. Extraction of microbial populations from plant tissue Foliar samples of approximately 8 g fresh weight of the youngest fully expanded compound leaves were
49 collected aseptically into sterile plastic bags. Microorganisms associated with the foliage were sampled following the methods of Heuer and Smalla (1999). Leaves were suspended in 20 mL sterile phosphate buffered saline (pH 7.2, 100 mmol L) before processing in a stomacher blender. Plant debris was removed from extracts by low speed centrifugation (5 min, 500 × g, 20 ◦ C). Dilutions of extracts were immediately plated for enumeration of microbial populations, as described below. Root samples consisted of 5 g (fresh weight) of roots with adhering soil. Roots were shaken to remove loosely attached soil before weighing into sterile stomacher bags for extraction by the method described by Heuer et al. (2002). Samples were extracted three times for 1 min in a stomacher blender with sterile buffer solutions. The three extracts were then combined and 1 mL was used for serial dilution and plating. Tubers were sampled by peeling the surface layer of tuber (approximately 5 g) and extracting as for root samples. Enumeration of culturable bacterial and fungal populations The leaf or soil extracts of each sample were serially diluted and plated on various agars to enumerate microbial populations. Total culturable bacteria were enumerated on 0.1 strength Tryptic Soy Agar (TSA: 3 g/L, Gibco BRL) containing 100 mg/L cycloheximide. Gram-negative bacteria were enumerated on Eosin Methylene-blue Lactose Sucrose Agar (EMB, Fort Richard) while Gram-positives were counted on Columbia CNA Sheep Blood Agar (Fort Richard). Pseudomonads were enumerated on Pseudomonas Isolation Agar (Difco). Bacterial colony forming units (cfu) were counted after 48 h incubation at 20 ◦ C. Selectivity of the agars was confirmed by subsequent identification of colonies, randomly selected from each of the agars. Total culturable fungi from leaf, root and tuber extractions were enumerated on potato dextrose agar (PDA; Merck) containing chlorotetracycline (10 µg/L). Yeasts from root and tuber samples were also enumerated on Dichloran Rose Bengal Chloramphenicol agar (DRBC) (Atlas, 1997). Endophytic fungi Separated leaf, root and tuber tissue was surface sterilised by soaking in 2% sodium hypochlorite for 5 min, rinsed in sterile distilled water and air dried on sterile
filter paper. Excised pieces (4–6 mm) of surface sterilised plant tissue were placed on water agar containing chlorotetracycline (10 µg/L). Plates were incubated at 20 ◦ C for 7–21 days before colony forming units were enumerated. Identification of bacterial and fungal isolates Bacterial isolates were randomly selected from TSA plates used for enumeration of bacterial populations on the tubers. Colonies were streaked for purification and Gram-stained. Gram-negative isolates were identified using commercial identification kits (API, bioMerieux). Fungal isolates recovered on PDA were identified by direct light microscope examination or sub-cultured onto Potato-carrot agar and Malt extract agar (Gams et al., 1987) and incubated at 20 ◦ C until sporulation. Cultures were identified to genus/species level on the basis of morphological characteristics (Watanabe, 2002; Gams et al., 1987). In vitro assay of magainin against rhizobacteria Susceptibility of bacterial isolates collected from tuber surfaces to synthetic magainins was assessed using a method adapted from Wilson and Conner (1995). Two antimicrobial peptides, magainin II and Ala-magainin (Sigma Chemical Co, USA) were used in assays, as these two forms most closely represented the magainin peptide expressed in plants (Barrell, 2001). A preliminary assay was conducted before testing of rhizobacteria with magainin analogues. E. carotovora (strain AgRB2447, held in the AgResearch culture collection, Lincoln New Zealand) was grown in the presence of a range of doses of the two peptides. The strain was more sensitive to Ala-magainin than magainin II, for which a dose exceeding 100 µg/mL was necessary to cause significant disruption of growth in broth. Test strains of rhizobacteria were grown overnight in tryptic soy broth (TSB) (Difco) and 50 µL of overnight culture (containing approximately 2×109 cfu/mL) was used to inoculate 950 µL TSB containing 100 µg/mL magainin II or 50 µg/mL Ala-magainin. Cultures were incubated for 24 h at 24 ◦ C on an Eppendorf Thermomixer (400 rpm) before being dilution plated on Tryptic Soy agar (Difco). Colonies were counted after 3 days incubation at 24 ◦ C and numbers of cfu/mL were compared with counts from control tubes for each isolate.
50 Statistical analysis Numbers of colony forming units/g plant material (fresh weight) were log10 transformed before analysis of variance was conducted. Comparison of the variance in estimates from single and composite samples was conducted. However, because of the limited number of replicates available for sampling in the trial, bacterial and fungal counts from both the composite and single samples were pooled before comparisons between plant lines were conducted. Counts on bacteria and fungi on D2 and D3 lines were excluded from the comparison of effects between the plant lines as only one plot was sampled for each of these lines. Fungal endophyte populations were analysed on the basis of type (genus/species) and number of fungal colonies recorded growing on each plate, in which each plate contained the same number of plant tissue pieces. Bacterial counts (cfu/mL) from in vitro sensitivity tests were log10 transformed before comparison of treatments with control using analysis of variance. Counts of Gram-negative and Gram-positive bacteria were analysed separately as different numbers of each type were tested against the magainin analogues. Results Culturable bacterial populations associated with potato plants Mean numbers of aerobic culturable bacteria/g plant tissue estimated from single or composite samples did not differ significantly (P > 0.05) and bacterial counts from both the composite and single samples were pooled before analysis. In general, however, the variability was lower where composite samples were used. Aerobic culturable bacteria that could be recovered from the potato phyllosphere on TSA (at 11 weeks after planting) were between log10 5.6 and 6 cfu/g leaf tissue (fresh weight) (Figure 1). Numbers of Gram-positive bacteria recovered from the leaves were slightly higher than Gram-negatives but this difference was not significant. Total numbers recovered did not vary significantly between the three plant lines tested. Similarly, there were no significant differences in the numbers of Gram- positive, Gram-negative and pseudomonads recovered from leaves collected from the three plant lines (Figure 1). Roots and tubers were sampled only at the end of the growing season when plants were senescent
Figure 1. Mean numbers of culturable bacteria in the phyllosphere of field grown potato plants. Log10 of colony forming units (cfu) per gram of leaf (fresh weight) for three plant lines: Karaka (unrelated cultivar), Iwa (non transgenic control), and D1 (transgenic test line). No significant differences were detected between the three plant lines (P > 0.05).
and tubers were fully developed. The total number of bacteria recovered from the roots was ca. log10 8.3 cfu/g root (fresh weight), with slightly less Grampositive than Gram-negative bacteria recovered, in contrast with the leaves (Figure 2A). The total number of culturable bacteria that could be recovered from the surfaces of roots did not differ significantly between the non transgenic control line and the transgenic test line, D1. Similarly, there were no significant differences between the various bacterial groups enumerated on selective agars. In contrast, numbers of Gram-positive bacteria were significantly lower on the surfaces of tubers from the transgenic test line D1, than in the non transgenic control line Iwa (P < 0.01) (Figure 2B). This difference was reflected in significantly lower total numbers of culturable bacteria being recovered from the transgenic line (P < 0.05). The number of culturable bacteria recovered from the rhizosphere (ca. log10 8.2 cfu/g) was larger than from the geocaulosphere (tuber surface) (ca. log10 6.9 cfu/g), as was also reported Lottmann et al. (1999). Culturable fungal populations associated with potato plants Culturable fungal populations associated with potato leaves did not vary significantly between the three plant lines tested, with ca. log10 5 cfu/g leaf. Both fungi and yeasts were enumerated from root and tuber surfaces (Figure 3). While yeast populations were
51
Figure 3. Mean number of culturable fungi in the phylloplane, and fungi and yeasts on the surfaces or roots and tubers of field grown potato plants. Log10 of colony forming units (cfu) per gram of leaf (fresh weight) for three plant lines: Karaka (unrelated cultivar), Iwa (non transgenic control), and D1 (transgenic test line). ∗ P < 0.05; ∗∗ P < 0.01; NS P > 0.05.
Figure 2. Mean numbers of culturable bacteria on the surface of A) roots and B) tubers of field grown potato plants. Log10 of colony forming units (cfu) per gram of leaf (fresh weight) for two plant lines: Iwa (non transgenic control), and D1 (transgenic test line). ∗ P < 0.05; ∗∗ P < 0.01; NS P > 0.05.
For roots, the numbers of cfu per plate varied between 0 and 12, with an overall mean of 4.8. The diversity of root endophytes (per plate) was similar to that for leaves, with up to 6 different species in a plate, and an overall mean of 2.5 different species per plate. For tubers, 82% of plates had no endophytes, and only 18% contained more than one colony. Because of the low numbers of colonies recovered, data on numbers of endophytes on tubers was not formally analysed but identities of colonies recovered are recorded in Table 1. Biodiversity of fungi associated with potato
not significantly different between the plant lines, the numbers of fungi recovered from the roots of the transgenic line were significantly higher than on the non transgenic control line (P < 0.05) (Figure 3). Endophytic fungi associated with potato plants The total number of cfu found per plate (leaves, roots) or per piece (tubers) was recorded. For leaves, total cfu varied between 0 and 6 per leaf, with an overall mean of 2.8. Up to six different endophyte species were found per leaf, with an overall mean of 2.2 different species on a leaf. Some differences were found between the plant lines (P < 0.05), with significantly more endophytes per leaf present for the D3 line (3.7) than for Iwa (2.5), Karaka (2.7) or D2 (2.2). D1 (3.1) was intermediate between D3 and the other plant lines.
Similar patterns of genera and species biodiversity were observed for both epiphytic and endophytic fungi isolated from the three tissue types (leaf, root, tuber) across all potato plant lines and the genera isolated are summarised in Table 1. Few endophytic fungi (12 spp.) were obtained from all the tuber pieces plated. Many of the fungal species encountered occurred sporadically and were not consistently associated with any particular plant line. However, not all viable species were identified, due to the diagnostic morphology not being produced in vitro or the isolates not matching existing taxonomic descriptions. The dominant epiphytic phylloplane fungi obtained across all plant lines were yeasts. The most frequently isolated filamentous fungi from all tissues and plant lines were either Hyphomycetous or Coelomycetous species. The potato phyl-
52 Table 1. Number of fungal species from each genus identified from potato plant lines in New Zealand
Fungal genus Acremonium Alternaria Aschochyta Aureobasidium Aspergillus Chaetomium Cladosporium Cylindrocarpon Colletotrichum Epicoccum Fusarium Gliocladium Humicola Microdochium Mortierella Paecilomyces Papulospora Penicillium Phoma Phomopsis Rhizoctonia Torula Trichoderma Verticillium Xylaria Other Total
Fungal classa H H C O H O H H C H H H H H O H H H C C O H H H O
Leaf Epiphytes 2 2 1 1
2
Leaf Endophytes 1 1 1
1 2
1 1 1 3
1 1
1 1 3
1 2
Root Epiphytes 3 1
2 2 1 2
6 3 1 1 4 3
1 1 1 1 1 2
3 3 1 1 2
17 42
1 9 32
2 1 1
Root Endophytes 5 1 1 1 1 2
Tuber Epiphytes
Tuber Endophytes
1 1 1 1 1 1
2 1 5 1 1 4 1 1 1 1 1 1
1 3
2
16 53
4 37
2 1 2 1 2 3
1 1
1
10 1 1 1 4 2 1 10 43
1
4 12
a Fungal class H = Hyphomycete C = Coelomycete O = Other (Agonomycete, Ascomycete, Basidiomycete, Zygomycete)
loplane was predominantly colonised by ubiquitous saprophytic airborne fungi such as Aureobasidium pullulans, Cladosporium cladosporioides and Epicoccum nigrum (Table 1). Common soilborne and root colonising fungi such as Cylindrocarpon, Fusarium, Paecilomyces, Penicillium and Trichoderma species were frequently isolated from both tuber and root pieces regardless of plant line, again the majority of these species are common saprophytes. All plant samples contained similar levels of sterile mycelial isolates that were classed as unidentified fungi. Further methods are required to determine taxonomic classification for such cryptic fungi. Surface sterilisation of plant tissue and antibiotic isolation media meant several classes of culturable fungi, such as Oomycetous species, were not obtained in the survey.
A minority of the fungal species observed have been previously recorded as being pathogenic to potato or related Solanum species (Table 2) either in New Zealand (Pennycook, 1989) or overseas (Farr et al., 1989). Again no difference was observed between the occurrences of these potentially pathogenic species in the various plant lines. In vitro assay of magainin against rhizobacteria A total of 34 bacterial isolates randomly collected from the surfaces of tubers (both D1 and Iwa lines) were assayed for sensitivity to two magainin analogues in vitro. A range of sensitivity patterns were observed (Figure 4), with some isolates showing sensitivity to both peptides, while a few were unaffected by the presence of magainin in the growth medium. Isolates often showed a higher level of sensitivity
53
Figure 4. Effect of magainin II (100 µg/mL) and Ala-magainin (50 µg/mL) in in vitro tests against A) Gram positive and B) Gram negative rhizobacteria where Isolates 1, 5 and 13 = Sphingomonas paucimobilis; 2 and 15 = Pseudomonas fluorescens; 3, 10, 14 and 16 = Sphingobacterium multivorum; 4 and 18 = Agrobacterium radiobacter; 6 = Sphingobacterium multivorum; 7, 11 and 22 = Pseudomonas sp.; 8 = Burkholderia sp.; 9 = Sphingobacterium spiritivorum; 12 and 19 = Stenotrophomonas maltophila; 17 and 21 = Chryseobacterium indologenes; 20 and 23 = Ralstonia pickettii; Erwinia = Erwinia carotovora strain AgRB2447.
to Ala-magainin than magainin II, despite the latter peptide being used at a higher rate. Log10 numbers of cfu/mL were compared with counts from the control tubes containing no magainin. Gram-negative and Gram-positive bacteria were analysed separately. For Gram-negative isolates (n = 23), both maginin II and Ala-magainin resulted in signi-
ficantly lower counts (P < 0.001) than the control (means of log10 7.95, 5.69 and 9.35, respectively). Counts from Ala-magainin treated broths were also significantly lower (P < 0.001) than those containing maginin II. For Gram-positive bacteria (n = 11), both magainin II and Ala-magainin resulted in significantly lower numbers of cfu than in the control
54 Table 2. Previously recorded fungal plant pathogens of Solanum tuberosum isolated from sampled tissues Fungal pathogen
Common name
Alternaria solani a Cercospora sp. b Colletotrichum sp. a Cylindrocarpon sp. b Doratomyces sp. b Fusarium spp. a Fusarium oxysporum a Mycosphaerella solani a Penicillium spp. b Rhizoctonia solani a Sclerotium sp. a Trichothecium roseum b Verticillium spp. a Verticillium dahliae a Xylaria sp. a
leaf spot leaf spot anthracnose root rot speck rot tuber, root rot, wilt wilt leaf spot blue mould rot damping off, rot, scab blight pink potato rot wilt wilt tuber rot
a previously recorded on S. tuberosum in New Zealand in Pennycook (1989). b previously recorded on S. tuberosum in North America in Farr et al. (1989).
(means of log10 6.65, 5.61 and 8.75, respectively). Gram-negative isolates were slightly more sensitive to Ala-magainin than magainin II but this difference was only significant at the 10% level. There was a significant interaction between sensitivity to magainin and the tuber type, with Gram-positive isolates recovered from D1 tubers giving higher numbers of cfu/mL in the presence of magainin II, than isolates from Iwa tubers (log10 8.21 cf. 5.76 cfu/mL).
Discussion Only a limited number of studies on the impact of transgenic plants on their associated microflora have been published to date and even fewer studies have been carried out using field grown plants. The present investigation examines the bacterial and fungal communities associated with magainin-expressing potato plants and unmodified control plants grown in the field. Using western analysis (Barrell et al. 2003), expression of magainin has been shown in extracts from leaves (Barrell, 2001) and from field- grown roots (Conner et al., unpublished results), but this method has not yet been used with tubers and levels of expression of magainin in these plant tissues remain unknown. However, bioassays showed that the transgenic tubers were more resistant to infection by
E. carotovora than unmodified control tubers (Barrell, 2001). If there is any effect of magainin-expressing potato plants on non-target organisms, it would most likely be detected upon investigation of root- and tuber-associated bacteria. Viable plating on selective agars was conducted to quantify various components of the bacterial communities. While this approach examined only culturable bacteria, it provided an opportunity to identify species and develop a collection of representative strains for further in vitro tests on sensitivity of phylloplane and rhizosphere isolates for sensitivity to magainin. The results of the in vitro sensitivity tests indicate that many of the bacteria commonly found on the surfaces of tubers could potentially be affected if magainin was secreted at significant level into the surrounding soil. Levels of magainin peptides used in in vitro experiments may well be higher than those expressed by the plants and probably represent a ‘worst case scenario’ in terms of potential effects on potatoassociated rhizobacteria. However, they are similar to rates used in tests against clinical isolates of bacteria (e.g., Giacometti et al., 1998) and in tests against bacterial and fungal plant pathogens (Alan and Earle, 2002). In a previous study looking at the sensitivity of clinical isolates to magainin II, sensitivity within the Gram-negative bacteria varied widely; the peptide was effective against almost all members of the Enterobacteriaceae tested but had little effect on three Pseudomonas species tested (Giacometti et al., 1998). Pseudomonas isolates tested in this study varied in sensitivity to the magainin analogues, with one isolate showing no sensitivity to either peptide (Isolate 7 in Figure 4B), while others showed a high level of sensitivity to both peptides (e.g., Isolate 22). Pseudomonads are among the most common bacteria found associated with plants and many are beneficial to plants, having plant growth promotion and/or biocontrol capabilities. No significant differences were found in total numbers of pseudomonads recovered from leaves, roots or tubers of magainin-expressing plants in this study, compared to the parental line. However, further testing of larger numbers of Pseudomonas isolates is warranted. The total culturable bacterial counts recovered in the rhizosphere (ca. log10 8.2 cfu/g root and log10 6.9 cfu/g tuber) were comparable to those found in other studies looking at a range of host plants, including potato (e.g., Lottman et al., 1999). While it is known that only some bacteria can be recovered and enumerated on culture media, this methodology
55 remains useful, despite its limitations. The sensitivity of analysis of viable counts depends on the level of variability which comes from several sources, including natural variability in microbial populations. Some of this variability can be accounted for by use of appropriate sampling methods. Heuer and Smalla (1999) used composite samples of potato foliage because the high variability of bacterial communities impeded a comparison between plant cultivars. However, even composite sampling of similarly aged leaves from several plants did not overcome the problems of aggregated distribution of certain bacteria. Because of these difficulties, it is generally recommended that several methods should be used in analysis of plant associated microbial communities. A clear limitation of the methodology used in this study was that only culturable bacteria were investigated and future studies on magainin-expressing potatoes will include molecular analysis of the bacterial communities by the culture-independent method, denaturing gradient gel electrophoresis (DGGE). Several studies using DGGE to analyse bacterial communities associated with transgenic plants have recently been conducted (e.g., Heuer and Smalla, 1999; Heuer et al., 2002). Because DGGE profiles are derived independently of cultivation, they more accurately reflect abundant species and provide a more complete profile of the microbial community. While bacterial communities associated with potatoes have been studied recently (e.g., Krechel et al., 2002), few studies have examined the fungal microflora associated with potato plants. Several studies have found that selected species of yeasts and filamentous fungi are potentially sensitive to environmental conditions as well as cultural practices (Edwards et al., 1996; van Bruggen and Semenov, 2000). Studies on the population dynamics of fungi have shown environmental and biotic disturbances can alter population patterns of phylloplane fungi (Waipara et al., 2002; Pennycook and Newhook, 1981) as well as root and soil-colonising fungi (Waipara, 2002). This study provided an opportunity to determine if similar effects could be measured in fungal populations associated with transgenic and non modified potato lines. Fungi associated with the potato phyllosphere have been investigated infrequently (e.g., Hollomon, 1967) while fungal communities associated with potato roots and tubers remain unknown. This study found that a similar species biodiversity was observed across all potato plant lines but some of the fungi isolated differed according to the type of plant tissue sampled.
All phylloplane samples were colonised by ubiquitous airborne species while Coelomycetous species were the most frequently obtained leaf endophytic fungi. Tuber and root tissues were most frequently colonised by previously described soilborne fungi that are present in most temperate soils or plant root tissues throughout the world (Domsch et al., 1980). These results are consistent with similar mycological surveys undertaken for other plant species in New Zealand (Skipp and Christensen 1981, 1989). Previous studies have shown plant pathogenic fungi have been affected by exposure to magainin peptides (Kristyanne et al., 1997). In particular the growth of soil-borne species Fusarium oxysporum, Rhizoctonia solani and Verticillium dahliae, which were encountered in this study from both transgenic and non transgenic plant tissues, was adversely affected through degradation of fungal organelles and cells. Spore germination of fungal tree pathogens was also inhibited after in vitro exposure to magainin II peptides (Jacobi et al., 2000). While magainin peptides may potentially enable some plant hosts to resist deleterious fungal infection, further investigation of the effects on beneficial and saprophytic fungal species should be conducted as these non-target species comprised the majority of culturable microfungi colonising potato plant tissues. This aspect will be investigated in future research undertaken to elucidate population dynamics of saprophytic and pathogenic fungi associated with these potato plant lines. Further research and methods are required to elucidate the biodiversity of non-culturable fungi, sterile mycelium and in particular the yeast populations present, as yeast populations have previously been found to be highly sensitive to environmental changes (Pennycook and Newhook, 1981; di Menna, 1966). Such studies will enable an increased understanding of the fungal populations associated with S. tuberosum, including both transgenic and non-transgenic lines.
Acknowledgements We are grateful to Friday Obanor, HortResearch, Lincoln for assistance in the laboratory and Jill Reader, Crop and Food Research, Lincoln for maintaining the field trial. Dave Saville, AgResearch, Lincoln and Ruth Butler, Crop and Food Research, Lincoln, carried out the statistical analyses.
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